System and method for processing and detecting nucleic acids

ABSTRACT

A system and method for processing and detecting nucleic acids from a set of biological samples, comprising: a capture plate and a capture plate module configured to facilitate binding of nucleic acids within the set of biological samples to magnetic beads; a molecular diagnostic module configured to receive nucleic acids bound to magnetic beads, isolate nucleic acids, and analyze nucleic acids, comprising a cartridge receiving module, a heating/cooling subsystem and a magnet configured to facilitate isolation of nucleic acids, a valve actuation subsystem configured to control fluid flow through a microfluidic cartridge for processing nucleic acids, and an optical subsystem for analysis of nucleic acids; a fluid handling system configured to deliver samples and reagents to components of the system to facilitate molecular diagnostic protocols; and an assay strip configured to combine nucleic acid samples with molecular diagnostic reagents for analysis of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/795,003, filed 19 Feb. 2020, which is a continuation of U.S.application Ser. No. 16/794,928, filed 19 Feb. 2020, which is acontinuation-in-part of U.S. application Ser. No. 15/996,949, filed 4Jun. 2018, which is a continuation of U.S. application Ser. No.15/413,735, filed 24 Jan. 2017, which is a continuation of U.S.application Ser. No. 14/613,616, filed 4 Feb. 2015, which is acontinuation-in-part application of U.S. application Ser. No. 13/766,359filed on 13 Feb. 2013, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/667,606, filed on 3 Jul. 2012, and U.S.Provisional Application Ser. No. 61/598,240, filed on 13 Feb. 2012,which are each incorporated herein in their entirety by this reference.This application is also related to U.S. application Ser. No.13/765,996, which is incorporated herein in its entirety by thisreference.

U.S. application Ser. No. 16/794,928, filed 19 Feb. 2020, is acontinuation of U.S. application Ser. No. 15/996,949, filed 4 Jun. 2018,which is a continuation of U.S. application Ser. No. 15/413,735, filed24 Jan. 2017, which is a continuation of U.S. application Ser. No.14/613,616, filed 4 Feb. 2015, also claims the benefit of U.S.Provisional Application Ser. No. 62/065,500, which are each incorporatedherein in their entirety by this reference.

TECHNICAL FIELD

This invention relates generally to the molecular diagnostics field, andmore specifically to an improved system and method for processing anddetecting nucleic acids.

BACKGROUND

Molecular diagnostics is a clinical laboratory discipline that hasdeveloped rapidly during the last 25 years. It originated from basicbiochemistry and molecular biology research procedures, but now hasbecome an independent discipline focused on routine analysis of nucleicacids (NAs), including deoxyribonucleic acids (DNAs) and ribonucleicacids (RNAs) for diagnostic use in healthcare and other fields involvinganalysis of nucleic acids. Molecular diagnostic analysis of biologicalsamples can include the detection of one or more nucleic acid materialspresent in the specimen. The particular analysis performed may bequalitative and/or quantitative. Methods of analysis typically involveisolation, purification, and amplification of nucleic acid materials,and polymerase chain reaction (PCR) is a common technique used toamplify nucleic acids. Often, a nucleic acid sample to be analyzed isobtained in insufficient quantity, quality, and/or purity, hindering arobust implementation of a diagnostic technique. Current sampleprocessing methods and molecular diagnostic techniques are oftenlabor/time intensive, low throughput, and expensive, and systems ofanalysis are insufficient. Furthermore, pre-analytical, analytical, andpost-analytical methods of isolation, processing, and amplification arespecific to certain sample matrices and/or nucleic acid types and notapplicable across common sample and nucleic acid types.

Due to these and other deficiencies of current molecular diagnosticsystems and methods, there is thus a need for an improved system andmethod for processing and detecting nucleic acids. This inventionprovides such a system and method.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict an embodiment of a system for processing anddetecting nucleic acids;

FIGS. 2A-2B depict an embodiment of elements, and a top view of anembodiment of a system worktable, respectively, of an embodiment of asystem for processing and detecting nucleic acids;

FIGS. 3A-3B depict an embodiment of a capture plate for combining asample with magnetic beads;

FIG. 4 depicts an embodiment of a capture plate module to facilitatelysis of a biological sample and combination of the biological samplewith magnetic beads;

FIGS. 5A-5B depict an alternative embodiment of a capture plate;

FIGS. 6A-6B depict embodiments of a molecular diagnostic module forprocessing and detecting nucleic acids;

FIGS. 7A-7E depict a sequence of operations performed by elements of anembodiment of a molecular diagnostic module;

FIG. 8 depicts an embodiment of a microfluidic cartridge and anembodiment of a cartridge platform;

FIGS. 9A-9B depict configurations of a linear actuator of an embodimentof a molecular diagnostic module;

FIGS. 9C-9E depict configurations of a portion of an embodiment of amicrofluidic cartridge and a molecular diagnostic module;

FIGS. 10A-10B depict elements of an embodiment of a valve actuationsubsystem of a molecular diagnostic module;

FIGS. 11A-11C depict an embodiment of a valve actuation subsystem of amolecular diagnostic module;

FIGS. 12A-12D depict elements of an embodiment of an optical subsystemof a molecular diagnostic module;

FIG. 13 depicts a side view of an alternative embodiment of a moleculardiagnostic module for processing and detecting nucleic acids;

FIGS. 14A-14C depict an embodiment of a fluid handling system of asystem for processing and detecting nucleic acids;

FIG. 15 depicts embodiments of elements of the fluid handling system;

FIGS. 16A-16C are schematics depicting example methods for processingand detecting nucleic acids;

FIGS. 17A-17B show embodiments of consumables and reagents used in asystem for processing and detecting nucleic acids;

FIGS. 18A-18B depict an embodiment of an assay strip to facilitateanalysis of a sample containing nucleic acids;

FIG. 19 depicts an embodiment of an assay strip holder;

FIG. 20 depicts an embodiment of an assay strip carrier;

FIGS. 21A-21B show alternative embodiments of assay strip holders andassay strips, respectively;

FIG. 22 shows an embodiment of a filter to facilitate processing anddetecting of nucleic acids;

FIG. 23 shows an embodiment of a filter holder to facilitate processingand detecting of nucleic acids;

FIGS. 24A-24D depict embodiments of a method for processing anddetecting nucleic acids; and

FIGS. 25A-25B depict embodiments of a method for processing anddetecting nucleic acids.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of preferred embodiments of the invention isnot intended to limit the invention to these preferred embodiments, butrather to enable any person skilled in the art to make and use thisinvention.

1. System for Processing and Detecting Nucleic Acids

As shown in FIGS. 1A-1B and 7A, an embodiment of a system 100 forprocessing and detecting nucleic acids comprises: a capture plate 110configured to facilitate binding of nucleic acids within a biologicalsample to a set of magnetic beads 119; a molecular diagnostic module 130comprising a microfluidic cartridge receiving module 140, heating andcooling subsystem 150, a magnet 160, a valve actuation subsystem 170, anoptical subsystem 180; and an assay strip 190 configured to facilitatemixing of molecular diagnostic reagents with a nucleic acid volume.Other embodiments of the system 100 may further comprise at least one ofa capture plate module 120 configured to support the capture plate 110;a filter 200 and filter holder 205 to facilitate sample preparation; amicrofluidic cartridge 210 configured to facilitate sample processing(e.g., where the cartridge does not contain reagents inside thecartridge prior to processing in the molecular diagnostic module, wherethe cartridge contains reagents); an assay strip holder 230; an assaystrip carrier 240; a liquid handling system 250 configured to facilitategas and fluid delivery to different elements of the system 100; aprocessor configured to analyze data resulting from a run of the system100; and a user interface configured to allow a user to interact withthe system 100. The system 100 thus functions to receive biologicalsamples containing nucleic acids (i.e., impure nucleic acid samples),separate nucleic acids from the biological samples, and analyze nucleicacid samples according to at least one molecular diagnostic protocol(e.g., PCR). Preferably, the system 100 is a walkaway system by which auser loads a set of biological samples containing nucleic acids, andreceives a set of data resulting from a molecular diagnostic protocolwithout any further sample manipulation by the user. Alternatively, thesystem 100 facilitates aspects of sample preparation for a moleculardiagnostic protocol, with some sample manipulation performed by theuser.

In one example workflow of the system 100, a liquid handling system 250aspirates a set of biological samples containing nucleic acids (i.e.,impure nucleic acid samples), and dispenses the set of biologicalsamples into a capture plate 110 to be lysed and combined with magneticbeads (containing a proprietary affinity coating to bind the nucleicacids to the magnetic beads) by a capture plate module 120. The liquidhandling system 250 then aspirates substantially all of each sample ofthe set of lysed biological samples combined with magnetic beads (i.e.,set of magnetic bead-samples) from the capture plate 110, and dispensesthe set of magnetic bead-samples into a microfluidic cartridge 210,aligned within a cartridge receiving module 140 of a moleculardiagnostic module 130, and configured to be manipulated by the moleculardiagnostic module 130. In particular, as shown in FIG. 2B, units of themicrofluidic cartridge 210 can be stored in a storage region 21 of thesystem. The system can further include a heating and cooling subsystem150, a magnet 160, and a valve actuation subsystem 170 of the moleculardiagnostic module 130 then facilitate separation of a set of nucleicacids from the magnetic bead-samples, as the liquid handling system 250dispenses wash solutions, release solutions, and/or air at appropriatestages. The liquid handling system 250 then aspirates the set of nucleicacids from the microfluidic cartridge 210 contained within the moleculardiagnostic module 130, combines the set of nucleic acids with a set ofmolecular diagnostic reagents using an assay strip 190, and dispensesthe set of nucleic acids combined with the set of molecular diagnosticreagents (i.e., set of nucleic acid-reagent mixtures) into themicrofluidic cartridge 210 within the molecular diagnostic module 130.The detection chamber heaters 157, optical subsystem 180 and valveactuation subsystem 170 of the molecular diagnostic module 130 thenfacilitate analysis of the set of nucleic acid-reagent mixtures by aprocessor configured to display information on a user interface.

As stated, the above workflow is just one example workflow of the system100, and other workflows of the system 100 and methods of processing anddetecting nucleic acid samples are further described in Section 2 below.A detailed description of elements of an embodiment of the system 100are described in sections 1.1-1.6 below.

1.1 System—Capture Plate and Capture Plate Module

As shown in FIGS. 3A and 3B, the capture plate 110 comprises a captureplate substrate 111 comprising a set of wells 112 and a puncturable foilseal 115, and functions to facilitate binding of nucleic acids within abiological sample to a set of magnetic beads 119. Preferably, the entirecapture plate 110 is configured to be a consumable (i.e., disposable),such that each well of the capture plate 110 can only be used once yetthe remaining unused wells can be used during additional runs of thesystem 100. Alternatively, at least a portion of the capture plate 110is configured to be reusable, such that additional mixing or reagentadditions can be performed and portions of the capture plate 110 may beused for multiple runs of the system 100. In one variation of thecapture plate 110, the capture plate substrate 111 is reusable, whilethe puncturable foil seal 115 is disposable and replaced after each runof the system 100.

The capture plate substrate 111 is configured such that the captureplate 110 is capable of resting on a flat surface, can be stacked withanother capture plate 110, and also can be manipulated with industrystandard instrument components for handling of microtiter plates. Thecapture plate substrate also functions to define the set of wells 112and to couple to the puncturable foil seal 115. The capture platesubstrate 111 is preferably composed of a PCR-compatible polymer thatcan be heat processed to couple to the puncturable foil seal 115, butcan alternatively be composed of any appropriate material that cancontain a fluid and be bonded to the puncturable foil seal 115.

The set of wells 112 of the capture plate substrate 111 function toreceive at least one biological sample which contain or are suspected ofpotentially containing nucleic acids, and to facilitate combination ofthe biological sample with a set of magnetic beads 119. Preferably, thewells 113 are each configured to accommodate not only a biologicalsample, but also to facilitate mixing of the biological sample with aset of magnetic beads 119 (e.g., using a pipettor, the liquid handlingsystem 250 or other apparatus), which preferably are preloaded in wells112, or alternatively may be added by an operator. Preferably, the wellsare also deeper than they are wide to allow a significant number ofwells 112 (e.g., 24) with clinically relevant sample volumes, and evenlyspaced to facilitate aspiration, delivery, and/or mixing of multiplebiological samples (e.g., with a multi-tip pipettor). Alternatively, thewells are wider than they are deep to facilitate larger devices formixing the biological samples with the magnetic beads 119. Each well 113of the set of wells 112 also preferably has a conically shaped bottomregion, as shown in FIG. 3A, to facilitate complete aspiration of afluid from a well. Alternatively, each well 113 may not have a conicallyshaped bottom region. Additionally, in the orientation shown in FIG. 3A,the tops of each well 113 in the set of wells 112 preferably form raisededges protruding from the capture plate substrate 111, in order tofacilitate sealing of each well 113 by the puncturable foil seal 115.Alternatively, the tops of each well 113 in the set of wells 112 may notform raised edges protruding from the capture plate substrate 111. Themagnetic beads are preferably polymer beads, precoupled with a ligandfor binding to a nucleic acid, and comprising a superparamagneticcomponent. Additionally, the magnetic beads may be treated to bepositively charged. However, the magnetic beads may alternatively be anyappropriate magnetic beads (e.g., magnetic, parmagnetic, orsuperparamagnetic) configured to facilitate biomagnetic separation.

Each quantity of magnetic beads 119 may be accompanied by lysingreagents (e.g. proteinase K) and a sample process control comprisingnucleic acid sequences for DNA and RNA, which function to lysebiological samples and to provide a mechanism by which sample processcontrols may be later detected to verify processing fidelity and assayaccuracy. The sample process control comprising nucleic acid sequencesfor DNA and RNA allows one version of the capture plate to facilitateassays involving DNA and RNA detection. Preferably, the quantity ofmagnetic beads 119, lysing reagents, and sample process controls isdried within each well to improve shelf life; however, the quantity ofmagnetic beads 119, lysing reagents, and sample process controls mayalternatively be in liquid form.

The puncturable foil seal 115 functions to isolate each well 113 of theset of wells 112, prevent contamination of the contents of each of theset of wells 112, protect the magnetic beads 119 and other reagentsstored in wells 112 from degradation, and provide informationidentifying the capture plate 110. The puncturable foil seal 115preferably seals each well 113 of the capture plate 110, and isconfigured to be punctured by an external element (e.g., by a pipettetip), such that each well is sealed prior to being punctured. In onevariation, the puncturable foil seal 115 also forms a seal around anelement that punctures it, and in another variation, the puncturablefoil seal 115 does not form a seal around an element that punctures it,in order to prevent airlock. The puncturable foil seal 115 is alsopreferably labeled with identifying information including at least oneof manufacturer information, capture plate contents, the lot of thecontents, an expiry date, and a unique electronic tag (e.g., barcode orQR code) providing more information. Preferably, the puncturable foilseal 115 does not extend beyond the footprint of the capture plate 110,but alternatively, the puncturable foil seal 115 may be any appropriatesize and/or include protruding features (e.g., tabs) that facilitatehandling of the capture plate.

In one variation, the capture plate 110 may be prepackaged at least withmagnetic beads 119, such that each well 113 in the set of wells 112 isprepackaged with a set of magnetic beads 119 defined by a specificquantity or concentration of magnetic beads. The set of wells 112 maythen be sealed by the puncturable foil seal 115, which is configured tobe punctured by an external element that delivers volumes of biologicalsamples to be mixed with the magnetic beads 119. In another variation,the capture plate 110 may not be prepackaged with magnetic beads 119,but the wells 113 of the capture plate may still be sealed with apuncturable foil seal 115. In this variation, the puncturable foil seal115 is configured to be punctured by at least one external element, forco-delivery of biological samples and magnetic beads intended to becombined.

A variation of the capture plate 110′ may further comprise a slottedrubber membrane 116, as shown in FIGS. 5A and 5B, configured to provideaccess through the puncturable foil seal 115 to the set of wells 112.The slotted rubber membrane 116 thus functions to prevent or reducesplashing, evaporation, and/or aerosolization of contents of the set ofwells 112. Preferably, the slotted rubber membrane 116 comprises slotsthat are self-sealing and centered over wells of the set of wells 112,and further does not extend beyond the footprint of the capture plate110. Alternatively, the slots of the slotted rubber membrane 116 may notbe self-sealing, and/or the slotted rubber membrane 116 may be anyappropriate size and comprise features that extend beyond the footprintof the capture plate 110.

In a specific example, the capture plate 110 comprises 24 wells 113 withan 18 mm center-to-center pitch, each well having a volumetric capacityof 2 mL, and is compliant with Society for Laboratory Automation andScreening (SLAS) standards. Each well 113 of the capture plate 110 inthe specific example is also prepackaged with a specified quantity ofmagnetic beads 119, and comprises a protruding top edge that is heatsealed to a puncturable foil seal. In addition, each well 113 alsocontains other reagents beneficial for processing and monitoring thesample, including proteinase K and one or more specific nucleic acidstands designed to serve as a process control. The specific example ofthe capture plate 110 can thus combine two groups of 12 biologicalsamples with magnetic beads. The capture plate 110 in the specificexample is produced by injection molding, has a footprint of 127.75mm×85.5 mm, and is composed of a PCR-compatible polypropylene basedpolymer with a high vapor barrier.

An embodiment of the system 100 may further comprise a capture platemodule 120, as shown in FIG. 4, which functions to receive, support, andheat a capture plate 110. The capture plate module 120 preferablycomprises a thermally conducting substrate 121 configured to cradle acapture plate 110, a capture plate heater 123, a capture plate receivingmodule 125, and a capture plate electronics module 127. Preferably, thecapture plate module 120 functions to facilitate lysis of a biologicalsample deposited into a well 113 of the capture plate, and to facilitatebinding of nucleic acids (i.e., within a lysed biological sample) to aquantity of magnetic beads 119 within a well 113 of the capture plate110. In a specific example, the capture plate module 120 has dimensionsof 108 mm×156 mm×45 mm and is configured to rest on a flat surface.

The thermally conducting substrate 121 is configured to cradle andsupport the capture plate 110, and functions to conduct heat to the setof wells 112 of the capture plate 110. Preferably, the thermallyconducting substrate 121 is also configured to reversibly couple to thecapture plate 110, and comprises a set of indentations 122 that encircleeach well 113 in the set of wells 112. In one variation, theindentations 122 completely conform to the external surface of each well113 of the capture plate 110, but in another variation, the indentations122 may encircle a portion of each well 113 of the capture plate 110.Additionally, the indentations 122 are preferably thermally conductingin order to conduct heat to the set of wells 112, and portions of thethermally conducting substrate 121 aside from the indentations 122 arecomposed of non-conducting, rigid material. Alternatively, the entirethermally conducting substrate 121 may be composed of a material that isthermally conducting.

The capture plate heater 123 is preferably coupled to the thermallyconducting substrate 121, and functions to transfer heat, through thethermally conducting substrate 121, to a well 113 of the capture plate110. The capture plate heater 123 preferably conforms to at least aportion of an indentation 122 of the thermally conducting substrate 121,to facilitate heat transfer through the indentation 122 to an individualwell 113 of the capture plate 110. In this variation, the capture plateheater 123 is one of a set of capture plate heaters 124, wherein eachcapture plate heater 123 in the set of capture plate heaters 124transfers heat to an individual well 113 of the set of wells 112 of thecapture plate 110. Alternatively, the capture plate heater 123 mayconform to portions of multiple indentations 122 of the thermallyconducting substrate 121, such that the capture plate heater 123 isconfigured to transfer heat to multiple wells 113 of the capture plate110. Preferably, the capture plate heater 123 is a resistance heater,but alternatively, the capture plate heater 123 may be a Peltier or anyappropriate heater configured to transfer heat to the capture plate 110.The capture plate heater 123 may also further couple to a heat sink.

The capture plate receiving module 125 comprises a capture plateactuation system 126 that functions to couple the capture plate module120 to a capture plate 110. As shown in FIG. 4, the capture plateactuation system 126 comprises a structural support with hinged grips128 and at least one capture plate module actuator 129. The captureplate module actuator 129 is preferably a push-type solenoid with aspring return, but may alternatively be any appropriate linear actuator,such as a hydraulic actuator. The structural support with hinged grips128 preferably couples to the capture plate heater 123 and houses thecapture plate module actuator 129, such that, in a first configuration,actuation of the capture plate module actuator 129 outwardly displacesthe hinged grips (allowing the capture plate module 120 to receive acapture plate 110), and in a second configuration, actuation of thecapture plate module actuator 129 inwardly displaces the hinged grips(allowing the capture plate module 120 to couple to the capture plate110). The structural support with hinged grips 128 may further comprisea textured and/or high-friction surface configured to grip a captureplate 110, but alternatively may not comprise a textured and/orhigh-friction surface.

The capture plate electronics module 127 is coupled to the capture plateheater 123 and the capture plate actuation system 126, and functions toenable control of the capture plate heater 123 and the capture plateactuation system 126. Preferably, the capture plate electronics module127 modulates an output of the capture plate heater 123, in order tocontrollably heat at least one well 113 of the capture plate 110.Additionally, the capture plate electronics module 127 preferablymodulates the capture plate actuation system 126, in order tocontrollably couple the capture plate module 120 to a capture plate 110.Preferably, the capture plate electronics module 127 is coupled to anexternal power supply, such that the capture plate module 120 does notinclude an integrated power supply; however, in alternative embodiments,the capture plate electronics module 127 may be coupled to a powersupply integrated with the capture plate module 120.

1.2 System—Molecular Diagnostic Module

As shown in FIGS. 6A and 6B, an embodiment of the molecular diagnosticmodule 130 of the system 100 includes a cartridge receiving module 140,a heating and cooling subsystem 150, a magnet 160, a valve actuationsubsystem 170, and an optical subsystem 180, and functions to manipulatea microfluidic cartridge 210 for processing of a biological samplecontaining nucleic acids. The molecular diagnostic module 130 ispreferably configured to operate in parallel with at least one othermolecular diagnostic module 130, such that multiple microfluidiccartridges 210 containing biological samples may be processedsimultaneously. In a first variation, the molecular diagnostic module130 is configured to be stackable with another molecular diagnosticmodule 130 in a manner that enables access to a microfluidic cartridge210 within each molecular diagnostic module 130; an example of the firstvariation is shown in FIG. 6B, where the molecular diagnostic modules130 are stacked in a staggered configuration. In the first variation,each molecular diagnostic module 130 may further comprise locking pinsor other appropriate mechanisms to couple the stacked moleculardiagnostic modules 130 together. In another variation, the moleculardiagnostic module 130 may not be configured to stack with anothermolecular diagnostic module, such that the molecular diagnostic modules130 are configured to rest side-by-side on the same plane. Elements ofan embodiment of the molecular diagnostic module 130 are furtherdescribed in sections 1.2.1 to 1.2.5 below, and the system can includeone or more units of the molecular diagnostic module as described belowand shown in the FIGURES.

1.2.1 Molecular Diagnostic Module—Cartridge Receiving Module

As shown in FIG. 9A, the cartridge receiving module 140 of the moleculardiagnostic module 130 comprises a cartridge platform 141 including acartridge loading guiderail 142, a cartridge stop 143, a magnetreceiving slot 144, and a set of valve actuation slots 145; a linearactuator 146 configured to displace a microfluidic cartridge 210 restingon the cartridge platform 141; and a set of springs 148 coupled to thecartridge platform 141. The cartridge receiving module 140 thusfunctions to receive, align, and compress a microfluidic cartridge 210for processing of a biological sample according to a moleculardiagnostic assay protocol. As shown in FIGS. 7A-7C, the cartridgeplatform 141 is preferably configured to receive a microfluidiccartridge 210 along a cartridge loading guiderail 142 until it reaches acartridge stop 143, and be vertically displaced by the linear actuator146, which places a biasing force against the set of springs 148 coupledto the cartridge platform 141. The magnet receiving slot 144 and the setof valve actuation slots 145 provide access, by a magnet 160 and a valveactuation subsystem 170, to the microfluidic cartridge 210, as themicrofluidic cartridge is vertically displaced by the linear actuator146.

The cartridge platform 141 provides a cartridge location with respect tothe molecular diagnostic module and includes one or more of a cartridgeloading guiderail 142, a cartridge stop 143, a magnet receiving slot144, and a set of valve actuation slots 145, and functions to receiveand align a microfluidic cartridge 210, while providing access to themicrofluidic cartridge 210 by a magnet 160 and a valve actuationsubsystem 170. As shown in FIG. 8, an embodiment of the cartridgeplatform 141 includes a pair of parallel cartridge loading guiderails142, initiating at a pair of inwardly tapering protrusions configured toguide a microfluidic cartridge toward the pair of parallel cartridgeloading guiderails 142, and spanning two short edges of the cartridgeplatform 141. The embodiment of the cartridge platform 141 also includesa cartridge stop 143 comprising a vertical tab oriented perpendicular tothe cartridge loading guiderails 142, and spanning a long edge of thecartridge platform. Preferably, the cartridge loading guiderails 142 andthe cartridge stop 143 are configured such that a microfluidic cartridge210 slides between the cartridge loading guiderails 142 and hits thecartridge stop 143 to signal proper alignment. Alternatively, thecartridge loading guiderails 142 and the cartridge stop 143 may beconfigured such that a microfluidic cartridge slides over or along thecartridge loading guiderails 142, after which the cartridge stop 143couples to a portion of the microfluidic cartridge 210 to ensure properalignment of the microfluidic cartridge. Additional variations of thecartridge loading guiderails 142 and the cartridge stop 143 may be usedto enable reception and alignment of a microfluidic cartridge 210 by themolecular diagnostic module 130, and are known by those skilled in theart.

The embodiment of the cartridge platform 141 shown in FIG. 8 alsoincludes a set of valve actuation slots 145, oriented perpendicular tothe parallel cartridge loading guiderails 142 and configured to provideaccess to a valve actuation subsystem 170, and a magnet receiving slot144 located among the set of valve actuation slots 145. Preferably, themagnet receiving slot 144 and the set of valve actuation slots 145substantially span a long dimension of the cartridge platform 141, asshown in FIG. 8, and are configured to correspond to locations on amicrofluidic cartridge 210 requiring a magnetic field and/or valving toenable processing of a biological sample and nucleic acid detection oncethe microfluidic cartridge 210 has been aligned within the moleculardiagnostic module 130. Thus, alternative configurations of the magnetreceiving slot 144 and the set of valve actuation slots 145 mayaccommodate other cartridges with alternative regions requiring magneticfields and/or valving to enable other protocols. In one alternativeembodiment, the magnet receiving slot 144 and the set of valve actuationslots may comprise one continuous void of the cartridge platform 141,such that the cartridge platform 141 supports a microfluidic cartridge210 along the periphery of the microfluidic cartridge 210, but forms acontinuous void under a majority of the footprint of the microfluidiccartridge 210.

The linear actuator 146 functions to linearly displace a microfluidiccartridge 210 resting on the cartridge platform 141, in order tocompress the microfluidic cartridge 210 and position the microfluidiccartridge 210 between a cartridge heater 153 and an optical subsystem180 on one side of the microfluidic cartridge 210, and a magnet 160 anddetection chamber heaters 157 on another side of the microfluidiccartridge 210. The linear actuator 146 also functions to provide asufficient counterforce to the valve actuation subsystem 170 such that amicrofluidic cartridge 210 within the molecular diagnostic module 130remains properly situated upon manipulation by the valve actuationsubsystem 170. The linear actuator 146 further functions to move anozzle 149 coupled to the liquid handling system 250, in order to couplethe liquid handling system 250 to a fluid port 222 of the microfluidiccartridge 210. In the orientation of the molecular diagnostic module 130shown in FIGS. 7B and 7B, the linear actuator 146 is preferably coupledto a portion of the heating and cooling subsystem 150 a portion of theoptical subsystem 180, and the nozzle 149, and vertically displaces thecartridge heater 153, the optical subsystem 180, and the nozzle 149 toposition the cartridge heater 153, 180 and the nozzle 149 over themicrofluidic cartridge 210. The vertical displacement also allows themicrofluidic cartridge 210 to receive a magnet 160, which provides amagnetic field to facilitate a subset of a molecular diagnosticprotocol, and detection chamber heaters 157, which allows amplificationand/or detection of nucleic acids for molecular diagnostic protocolsrequiring heating and cooling of the nucleic acid (e.g. PCR).Preferably, the linear actuator 146 is a scissor jack actuatorconfigured to apply substantially uniform pressure over all occlusionpositions of a microfluidic cartridge 210 aligned within the moleculardiagnostic module 130, and to operate in at least two configurations. Ina retracted configuration 146 a, as shown in FIG. 9A, the scissor jackactuator has not linearly displaced the cartridge platform 141, and inan extended configuration 146 b, as shown in FIG. 9B, the scissor jackactuator has linearly displaced the microfluidic cartridge 210 toposition the microfluidic cartridge 210 between the subsystems 153, and180, and the magnet 160 and detection chamber heaters 157. Additionally,the extended configuration 146 b of the scissor jack actuator isconfigured to couple the nozzle 149 to a fluid port 222 of themicrofluidic cartridge 210, such that the liquid handling system 250 candeliver solutions and gases for processing of biological samples. Thelinear actuator 146 may alternatively be any appropriate linearactuator, such as a hydraulic, pneumatic, or motor-driven linearactuator, configured to linearly displace a microfluidic cartridgewithin the molecular diagnostic module 130.

As shown in FIGS. 7B, 7C, and 8, a set of springs 148 is coupled to thecartridge platform 141 and functions to provide a counteracting forceagainst the linear actuator 146 as the linear actuator 146 displaces amicrofluidic cartridge 210 resting on the cartridge platform 141. Theset of springs 148 thus allows the cartridge platform 141 to return to aposition that allows the microfluidic cartridge 210 to be loaded andunloaded from the molecular diagnostic module 130 when the linearactuator 146 is in a retracted configuration 146 b, as shown in FIG. 7B.Preferably, in the orientation shown in FIG. 7B, the set of springs 148is located at peripheral regions of the bottom side of the cartridgeplatform 141, such that the set of springs 148 does not interfere withthe magnet or the valve actuation subsystem 170. Alternatively, the setof springs 148 may be located at any appropriate position to provide acounteracting force against the linear actuator 146. In a specificexample shown in FIG. 6A, the set of springs 148 comprises four springslocated near corners of the bottom side of the cartridge platform 141,but in other variations, the set of springs 148 may comprise anyappropriate number of springs. Each spring of the set of springs 148 isalso preferably housed within a guide to prevent deviations from linearvertical motions (in the orientation shown in FIG. 7B); however, eachspring in the set of springs 148 may alternatively not be housed withina guide. In an alternative embodiment of the molecular diagnostic module130, the set of springs 148 may altogether be replaced by a secondlinear actuator configured to linearly displace a microfluidic cartridge210, resting on the cartridge platform 141, in a direction opposite tothe displacements enforced by the linear actuator 146, and/or by anyother suitable element (e.g., elastomeric element) configured to providea biasing force against a microfluidic cartridge 210 at the cartridgeplatform 141.

Similarly, the nozzle 149, the heating and cooling subsystem 150, thecartridge heater 153, and the magnet 160 are preferably coupled tosprings, such that springs are positioned between elements 149, 150,153, and 160, and substrates that elements 149, 150, 153, and 160 aremounted to. Alternatively an elastomeric material is preferablypositioned between elements 149, 150, 153, and 160, and substrates thatelements 149, 150, 153, and 160 are mounted to. The springs and/orelastomeric material function to provide proper functioning andalignment of subsystems of the molecular diagnostic module 130 as thelinear actuator 146 is extended or retracted, contributing toreliability and a reduction in stack up tolerance risk. The springsand/or elastomeric material further function to allow more pressure tobe applied to occlusion positions of a microfluidic cartridge 210aligned within the molecular diagnostic module 130, and an appropriatepressure to be applied to elements 149, 150, 153 and 160 of themolecular diagnostic module 130. Thus, proper contact is maintainedbetween elements 149, 150, 153, and 160, and a microfluidic cartridge210 being manipulated by the molecular diagnostic module. These elementsare described in further detail below.

1.2.2 Molecular Diagnostic Module—Heating/Cooling Subsystem and Magnet

The heating and cooling subsystem 150 of the molecular diagnostic module130 comprises a cartridge heater 153, a fan 155, and a set of detectionchamber heaters 157 and functions to controllably heat portions of amicrofluidic cartridge 210 for processing of a biological samplecontaining nucleic acids according to a molecular diagnostic protocol.In the orientation of an embodiment of the molecular diagnostic module130 shown in FIGS. 7A-7C, the cartridge heater 153 is preferably coupledto the linear actuator 146 of the cartridge receiving module 140 andconfigured to span a central region of a microfluidic cartridge 210aligned within the molecular diagnostic module 130, the fan 155 islocated at a back wall of the cartridge receiving module 140, and theset of detection chamber heaters 157 is located inferior to a set ofdetection chambers 213 of the microfluidic cartridge 210. In alternativeembodiments of the molecular diagnostic module 130, the heating andcooling subsystem 150 may have any appropriate alternative configurationthat provides controlled heating and cooling to a microfluidic cartridgewithin the molecular diagnostic module 130.

The cartridge heater 153 functions to transfer heat to a heating region224 of a microfluidic cartridge 210, for inducing a pH shift to releasebound nucleic acids from magnetic beads within the heating region 224.The cartridge heater 153 is preferably a plate-shaped heater configuredto transfer heat to the microfluidic cartridge 210 only from one side ofthe cartridge heater 153, such that heat flows through one face of theplate-shaped heater to the microfluidic cartridge 210. In a specificexample, the cartridge heater 153 is a silicon wafer etched to beconductive and form a resistance heater. In the preferred variation, thecartridge heater 153 is either flip-chip bonded (i.e., soldered to backside of a circuit board), or wire bonded to a circuit board, and thencoupled using linear bearings and springs to a plate coupled to thelinear actuator 146. The preferred variation allows independent controlof 12 independent channels, corresponding to 12 different pathways forsample processing. In another variation, heating through one face isaccomplished using a plate-shaped resistance heater that has one exposedface and thermal insulation covering all other faces, and in yet anothervariation heating through one face is accomplished using a Peltierheater. In a variation of the cartridge heater 153 using a Peltierheater, the cartridge heater 153 comprises a thermoelectric material,and produces different temperatures on opposite faces of the cartridgeheater 153 in response to a voltage difference placed across thethermoelectric material. Thus, when a current flows through the Peltierheater, one face of the Peltier heater lowers in temperature, andanother face of the Peltier heater increases in temperature. Alternativevariations of the cartridge heater 153 can be used to appropriatelytransfer heat to a heating region 224 of the microfluidic cartridge 210.

Preferably, the cartridge heater 153 is configured to linearly translatewith the linear actuator 146 of the cartridge receiving module 140, inorder to align with a heating region 224 spanning a central portion of amicrofluidic cartridge 210 aligned within the molecular diagnosticmodule 130. In one variation, the cartridge heater 153 is preferablyfixed relative to the linear actuator 146 such that (in the orientationshown in FIGS. 7B-7C), the cartridge heater 153 can only move verticallywith the linear actuator. In an alternative variation, the cartridgeheater 153 may additionally be configured to translate laterally with ahorizontal plane (in the orientation shown in FIGS. 7B-7C), such thatthe cartridge heater 153 can translate in at least two perpendicularcoordinate planes. In this alternative variation, the cartridge heater153 can be configured to sweep across a surface of a microfluidiccartridge 210 aligned within the molecular diagnostic module 130, or totranslate in response to motion of the microfluidic cartridge 210, suchthat the position of the cartridge heater 153 relative to a heatingregion 224 of the microfluidic cartridge 210 is always fixed.

The fan 155 functions to modulate heat control within the moleculardiagnostic module 130, by enabling heat transfer from warm objectswithin the molecular diagnostic module 130 to cooler air external to themolecular diagnostic module 130. In the orientation shown in FIG. 6A,the fan 155 is preferably located at a back face of the moleculardiagnostic module 130, such heat within the molecular diagnostic module130 is transferred out of the back face of the molecular diagnosticmodule 130 to cooler air external to the molecular diagnostic module. Ina specific embodiment, the molecular diagnostic module 130 comprisesfour fans 155 located at the back face of the molecular diagnosticmodule 130; however, in alternative embodiments the molecular diagnosticmodule 130 may comprise any appropriate number of fans located at anyappropriate position of the molecular diagnostic module 130. In onevariation, the fan 155 may be passive and driven solely by convectioncurrents resulting from motion of hot air within the moleculardiagnostic module to cooler air outside of the molecular diagnosticmodule; however, in alternative variations, the fan 155 may bemotor-driven and configured to actively cool internal components of themolecular diagnostic module 130 if molecular diagnostic module elementsexceed a certain threshold temperature.

The set of detection chamber heaters 157 functions to individually heatdetection chambers of a set of detection chambers 213 within amicrofluidic cartridge 210. Each detection chamber heater in the set ofdetection chamber heaters 157 is preferably configured to heat one sideof one detection chamber in the set of detection chambers 213, and ispreferably located such that the extended configuration 146 b of thelinear actuator 146 of the cartridge receiving module 140 puts adetection chamber in proximity to a detection chamber heater. Asmentioned above, the set of detection chamber heaters 157 is preferablycoupled to springs or an elastomeric layer to ensure direct contactbetween the set of detection chamber heaters and a set of detectionchambers, without compressively damaging the set of detection chamberheater 157. Preferably, each detection chamber heater is configured tocontact a surface of a detection chamber in the extended configuration146 b of the linear actuator 146; however, each detection chamber heatermay be further configured to couple to a detection chamber in theextended configuration 146 b of the linear actuator 146. In a firstvariation, the set of detection chamber heaters 157 comprises siliconchip heaters flip chipped to one surface of a flexible printed circuitboard, with a set of springs coupled to an opposite surface of theflexible printed circuit board, such that each spring in the set ofsprings aligns with a detection chamber heater. In the first variation,contact between each detection chamber heater and a detection chamber isthus maintained by a biasing force provided by an individual springthrough the flexible printed circuit board. In a second variation, theset of detection chamber heaters 157 comprises silicon chip heaters flipchipped to one surface of a rigid printed circuit board, with a set ofsprings coupled to an opposite surface of the rigid printed circuitboard. In the second variation, the set of springs thus function tocollectively transfer a force through the rigid printed circuit board tomaintain contact between the set of detection chamber heaters and a setof detection chambers. Preferably, each detection chamber heater in theset of detection chamber heaters 157 is configured to contact and heat abottom surface of a detection chamber (in the orientation shown in FIG.7B); however, each detection chamber heater may alternatively beconfigured to contact and heat both a top and a bottom surface of adetection chamber. Additionally, each detection chamber heaterpreferably corresponds to a specific detection chamber of the set ofdetection chambers 213 and functions to individually heat the specificdetection chamber; however, alternatively, each detection chamber heatermay be configured to heat multiple detection chambers in the set ofdetection chambers 213. Preferably, all detection chamber heaters in theset of detection chamber heaters 157 are identical; however, the set ofdetection chamber heaters 157 may alternatively not comprise identicaldetection chamber heaters.

In one variation, each detection chamber heater in the set of detectionchamber heaters 157 comprises a donut-shaped heater, configured toencircle a surface of a detection chamber. The donut-shaped heater mayfurther include a conducting mesh configured to allow detection throughthe heater while still allowing efficient heat transfer to the detectionchamber. In an alternative variation, each detection chamber heater inthe set of detection chamber heaters 157 may include a plate-shapedPeltier heater, similar to Peltier cartridge heater 153 described above.In this alternative variation, each detection chamber heater is thusconfigured to heat one side of a detection chamber through one face ofthe detection chamber heater. In one specific example, the moleculardiagnostic module 130 comprises 12 diced silicon wafers with conductivechannels flip chipped to 12 detection chambers, providing resistiveheating to each of the 12 detection chambers. In another specificexample, the molecular diagnostic module 130 comprises a 12 Peltierdetection chamber heaters configured to heat 12 detection chambers of amicrofluidic cartridge 210 aligned within the molecular diagnosticmodule 130. In other alternative variations, each detection chamberheater may comprise any appropriate heater configured to individuallyheat a detection chamber.

In some variations, reflection from the set of detection chamber heaters157 can interfere with light transmitted to photodetectors of theoptical subsystem 180 (e.g., light emitted from the set of biologicalsamples, light transmitted through filters of an optical subsystem),especially in configurations wherein the set of detection chambers 213of a microfluidic cartridge 210 are positioned between detection chamberheaters 157 and optical elements of an optical subsystem 180. In thesevariations, the set of detection chamber heaters 157 can includeelements that reduce or eliminate reflection from the set of detectionchamber heaters 157, thereby facilitating analysis of the set ofbiological samples. In one variation, the set of detection chamberheaters 157 can include or be coupled to non-reflective coatings atsurfaces of the set of detection chamber heaters 157 upon which lightfrom the optical subsystem 180 impinges. In a specific example, thenon-reflective coating can comprise a high-temperature paint (e.g., darkpaint, flat paint) that absorbs and/or diffuses light from the opticalsubsystem 180, while facilitating heat transfer to a set of detectionchambers 213 of a microfluidic cartridge 210. In another variation, theset of detection chamber heaters can be in misalignment withphotodetectors of the optical subsystem 180, such that reflection doesnot interfere with light transmitted to the photodetectors of theoptical subsystem 180. In one example, the set of detection chamberheaters can be configured to heat a set of detection chambers 213 from afirst direction, and the optical subsystem 180 can be configured toreceive light from the set of detection chambers 213 from a seconddirection (e.g., a direction non-parallel to the first direction), suchthat reflection from the detection chamber heaters 157 does not causeinterference. In still other variations, the set of detection chamberheaters 157 can include any other suitable elements (e.g., coatings,layers, etc.) and/or be configured in any other suitable manner thateliminates, prevents, or mitigates reflection from the set of detectionchamber heaters 157 from interfering with light transmitted tophotodetectors of the optical subsystem 180.

The magnet 160 of the molecular diagnostic module 130 functions toprovide a magnetic field for isolation and extraction of nucleic acidsbound to magnetic beads within a microfluidic cartridge 210, alignedwithin the molecular diagnostic module 130. Preferably, the magnet 160is fixed within the molecular diagnostic module 130, such that theextended configuration 146 b of the linear actuator 146 allows themagnet 160 to pass through the magnet receiving slot 144 of thecartridge receiving module 140 and into a magnet housing region 218 ofthe microfluidic cartridge 210. In an example, as shown in FIGS. 7A-7C,the magnet 160 is a rectangular prism-shaped magnet 160 fixed under thecartridge platform 141, and configured to pass through the cartridgeplatform 141, into a magnet housing region 218 located at a surface ofthe microfluidic cartridge 210 directly opposing the heating region 224of the microfluidic cartridge 210. As such, in some variations, aprojection of the heating region 224 onto a plane at least partiallyoverlaps with a projection of the magnet housing region 218 onto theplane. However, the magnet housing region 218 and the heating region 224of the microfluidic cartridge 210 can be in thermal communication in anyother suitable manner. Preferably, the magnet 160 is one of multiplemagnets (e.g., 2-3 magnets) lined up in parallel, such that each of thefluidic pathways of a microfluidic cartridge housing the magnets isexposed to a multiplied magnetic flux (e.g., two or three times as muchmagnetic flux), and a multiplied opportunity to capture magnetic beads.Alternatively, the magnet 160 is a single magnet configured to expose aset of fluidic pathways to a magnetic field. Preferably, the magnet 160or group of multiple magnets is coupled to a magnet holder within themolecular diagnostic module 130. Additionally, the magnet holder ispreferably composed of an insulating material, such that the magnetholder does not interfere with proper functioning of the cartridgeheater 153. Alternatively, the magnet holder may not be composed of aninsulating material.

In one variation, the magnet 160 or group of multiple magnets comprisesa permanent magnet, composed of a magnetized material (e.g., aferromagnet) providing a substantially fixed magnetic field. In analternative variation, the magnet 160 or group of multiple magnetscomprises an electromagnet configured to provide a modifiable magneticfield, such that the intensity of the magnetic field can be adjusted,the polarity of the magnetic field can be reversed, and the magneticfield can be substantially removed upon removal of a current flowingwithin the electromagnet. Preferably, the magnet 160 or group of magnetsis also fixed relative to the molecular diagnostic module 130; however,the magnet 160 or group of magnets may alternatively be configured totranslate vertically (in the orientation shown in FIG. 7B), such thatthe magnet 160 or group of magnets can extend into and retract from themagnet receiving slot 144 of the cartridge platform 141 and the magnethousing region 218 of the microfluidic cartridge 210. Additionally, themagnet 160 or group of magnets preferably rides on linear bearingsand/or springs (or an elastomeric material) to ensure proper contactwith a microfluidic cartridge in an extended configuration 146 b of thelinear actuator 146, in a manner that allows most of force from thelinear actuator 146 to translate to full occlusion of a subset of theset of occlusion positions (i.e., without leakage).

In some variations, wherein a magnet housing region 218 of themicrofluidic cartridge 210 is located at a surface of the microfluidiccartridge 210 directly opposing the heating region 224 of themicrofluidic cartridge 210, all or a subset of the magnet(s) 160 can beheated, such that the magnet(s) do not provide a heat sink at surfacesof the microfluidic cartridge 210 opposing the cartridge heater 153and/or any other portion of the microfluidic cartridge 210 intended tohave a desired heated state (e.g., a portion of the microfluidiccartridge proximal the set of detection chamber heaters 157).Preferably, heating of the magnet(s) 160 of the molecular diagnosticmodule is performed in a manner that does disrupt alignment of magneticdomains (e.g., for a permanent magnet), such that a magnetic fieldprovided by the magnet(s) 160 does not diminish in strength. As such, amagnet 160 of the molecular diagnostic module 130 is preferably heatedto a temperature less than its Curie point, or can additionally oralternatively comprise a magnetic material with a sufficiently highCurie point (e.g., a Curie point characterized by a higher temperaturethan temperatures required for processing of samples at the microfluidiccartridge 210). In one example, the magnet(s) 160 can thus be configuredto be heated to one or more temperatures in synchronization withtemperatures of the cartridge heater 153, in order to further increaseuniformity of heating through the microfluidic cartridge (e.g., from aheating region 224 to a magnet-housing region). The magnet 160 can,however, be any other suitable magnet (e.g., permanent magnet,electromagnet) that is not disrupted by heating within the range oftemperatures required for processing of samples at the microfluidiccartridge 210. Furthermore, the magnet(s) 160 of the moleculardiagnostic module 130 can be configured to be heated with any suitabletemperature output, such that the magnet(s) facilitate generation of anysuitable heating profile (e.g., non-uniform heating profile, uniformheating profile, etc.) through the microfluidic cartridge 210.

In one variation, the molecular diagnostic module 130 can comprise atleast one magnet 160 coupled to a magnet heating element, such that themagnet heating element heats the magnet 160 to a desired state. In oneexample of this variation, the molecular diagnostic module 130 cancomprise a set of magnets 260, wherein each magnet 160 of the set ofmagnets 260 is coupled to a magnet heating element 261 at least at onesurface of the magnet. As such, the magnet heating element can becoupled to a surface of the magnet 160, can wrap about multiple surfacesof the magnet, can be at least partially embedded in the magnet, and/orcan be coupled to the magnet in any other suitable manner. In onespecific example, as shown in FIG. 9E, each magnet 160 of the set ofmagnets 260 is separated from an adjacent magnet by a magnet heatingelement 261, and in another specific example, each magnet 160 of the setof magnets has a magnet heating element coupled to a distal end of themagnet 160, wherein the distal end of the magnet 160 is configured tointerface with the magnet housing region 218 of the microfluidiccartridge 210. The magnet(s) 160 and the magnet heating element(s) 261of the microfluidic cartridge 210 can, however, be configured in anyother suitable manner.

Additionally or alternatively, the molecular diagnostic module 130 canbe configured with an insulation gap between the magnet(s) 160 and asurface of the microfluidic cartridge 210 proximal the magnet housingregion 218, such that the magnet(s) 160 do not interfere with heating ofthe microfluidic cartridge 210. The insulation gap can be an air gapwithin the system or can additionally or alternatively comprise anyother suitable insulating layer situated between the magnet(s) 160 andthe surface of the microfluidic cartridge 210 opposing the cartridgeheater 153.

In any of the above embodiments and variations of the magnet(s), themagnet(s) are preferably configured to span a substantial portion of acapture segment 263 (e.g., an s-shaped capture segment with acharacteristic width) of the microfluidic cartridge 210, by way of themagnet housing region 218, wherein the capture segment is a portion of afluidic pathway configured to facilitate capture of target particlesbound to magnetic particles. As such, the magnet is preferablysubstantially wide in order to span a majority of the capture segmentand provide a desired gradient of magnetic strength at the capturesegment, by way of the magnet housing region 218 of the microfluidiccartridge 210. Additionally, the strength of the magnet(s) can beadjusted to prevent clogging within the capture segment, for instance,by adjusting morphology, composition, and/or any other suitablecharacteristic of the magnet(s). In one specific example, the magnet iswide enough to span a majority, but not all, of an s-shaped capturesegment 263 of a microfluidic cartridge, by crossing the s-shapedcapture segment in an orientation perpendicular to a flow directionthrough the s-shaped capture segment 163, as shown in FIGS. 9C-9E;however, the magnet(s) 160 can alternatively be configured in any othersuitable manner.

Alternative configurations and/or compositions of the magnet 160 mayalso be appropriate in facilitating isolation and extraction of nucleicacids bound to magnetic beads within the microfluidic cartridge 210. Assuch, the system can include an extraction subsystem configured toextract target content from the one or more samples, and a PCR operationsubsystem distinct from the extraction subsystem, wherein the extractionsubsystem is configured to perform one or more of: addition of anextraction buffer to the unit of the cartridge, a cell lysis operation,a protein degradation operation and nucleic acid isolation usingmagnetic particles.

1.2.3 Molecular Diagnostic Module—Valve Actuation Subsystem

As shown in FIGS. 10A-11C, the valve actuation subsystem 170 of themolecular diagnostic module 130 comprises a set of pins 172 configuredto translate linearly within a pin housing 175, by sliding a cam card177 laterally over the pins 172. The valve actuation subsystem 170functions to provide a biasing force to deform an object in contact withthe set of pins 172. In a configuration wherein a microfluidic cartridge210 is aligned within the molecular diagnostic module 130, the valveactuation subsystem 170 thus functions to occlude a fluidic pathway 220of the microfluidic cartridge 210 at a set of occlusion positions 226,to control flow of a biological sample containing nucleic acids,reagents and/or air through the microfluidic cartridge 210. In anembodiment of the molecular diagnostic module shown in FIGS. 7D-7E, theset of pins 172 and the pin housing are located directly under themicrofluidic cartridge 210, such that the set of pins can access themicrofluidic cartridge 210 through the valve actuation accommodatingslots 145 of the cartridge platform 141. The cam card 177 in theembodiment is positioned under the set of pins and is coupled to alinear cam card actuator 178 configured to laterally displace the camcard 177 to vertically displace pins of the set of pins 172. Preferably,as shown in FIG. 11A, the cam card 177 rests on a low friction surfaceconfigured to facilitate lateral displacement of the cam card 177;however, the cam card 177 may alternatively rest on a bed of ballbearings to facilitate lateral displacement of the cam card 177, or mayrest on any feature that allows the cam card 177 to be laterallydisplaced by the linear cam card actuator 178.

The cam card 177, as shown in FIGS. 7D and 11A, includes a set of hills176 and valleys 179, and functions to transform linear motion in oneplane to vertical motion in another plane. In one variation, the camcard 177 is coupled to a linear actuator and contacts the ends of pinsin a set of pins 172, such that when a hill 176 of the cam card 177passes under a pin, the pin is in a raised configuration 177 a, and whena valley 179 of the cam card 177 passes under a pin, the pin is in alowered configuration 177 b. The hills 176 and valleys 179 of the camcard 177 are preferably in a set configuration, as shown in FIG. 11B,such that lateral motion of the cam card 177 to a set position raises afixed subset of the set of pins 172. In this manner, lateral movement ofthe cam card 177 to different positions of a set of positionsconsistently raises different subsets of the set of pins 172 to occludedifferent portions of a fluidic pathway 220 of a microfluidic cartridge210 in contact with the set of pins 172. Thus, portions of a fluidicpathway 220 may be selectively occluded and opened to facilitateprocessing of a biological sample according to any appropriate tissue,cellular, or molecular diagnostic assay protocol. In one variation, thecam card is configured to be laterally displaced in two coordinatedirections within a plane (e.g., by x-y linear actuators), and inanother variation, the cam card is configured to be laterally displacedin only one coordinate direction within a plane (e.g., by a singlelinear actuator). In a specific example, the hills 176 of the cam card177 are raised 1 mm above the valleys 179 of the cam card 177, the hills176 and valleys 179 each have a 2 mm wide plateau region, and a hill 176region slopes down to a valley region 179 at a fixed angle over a 2 mmlength. In the specific example, the cam card 177 is driven by aFirgelli linear actuator. Alternative variations may include anyappropriate configurations and geometries of a cam card with hills 176and valleys 179, driven by any appropriate actuator.

In alternative embodiments of the valve actuation subsystem 170, the camcard 177 may be a cam card wheel comprising a set of hills 176 andvalleys 179 on a cylindrical surface, and configured to convert rotarymotion to linear (i.e., vertical) motion of the set of pins 172. The camcard wheel may be configured to contact ends of pins in the set of pins172, and may be coupled to a motor shaft and driven by a motor. In otheralternative embodiments of the valve actuation subsystem 170, the camcard 177 may altogether be replaced by a set of cams, each configured toindividually rotate about an axis. In these alternative embodiments,rotating subsets of the set of cams raises corresponding subsets of theset of pins, and occludes specific portions of a fluidic pathway 220 ofa microfluidic cartridge 210 in contact with the set of pins 172.

The set of pins 172 functions to selectively occlude portions of afluidic pathway 220 of a microfluidic cartridge 210 at least at subsetsof a set of occlusion positions 226. The pins of the set of pins 172 arepreferably cylindrical and, in the orientation shown in FIG. 11A,configured to slide over a cam card 177 and within a pin housing 175.Each pin in the set of pins 172 preferably also includes a first spring173 that functions to provide a counteracting force to restore a pin toa lowered configuration 177 b; however, each pin in the set of pins 172may alternative not include a first spring 173, and rely solely ongravity to return to a lowered configuration 177 b. Preferably, as shownin FIG. 11C, each pin is also composed of two parts separated by asecond spring, which functions to allow sufficient force to fullyocclude a microfluidic channel but prevents forces from being generatedthat could damage the pin, microfluidic cartridge and/or cam card. Eachpin also preferably comprises a first region 171 configured to slidewithin the pin housing 175, and a second region 174 configured to exitthe pin housing 175. The second region 174 is preferably of a smallerdimension than the first region 171, such that each pin is constrainedby the pin housing 175 to be raised by a limited amount. Alternatively,the first region 171 and the second region 174 may have any appropriateconfiguration to facilitate raising and lowering of a pin by a fixedamount. In a specific example, the valve actuation subsystem 170comprises 12 sets of pins 172 configured to selectively occlude 12fluidic pathways 212 of a microfluidic cartridge 210 aligned within themolecular diagnostic module; however, other embodiments may comprise anyappropriate number of sets of pins 172.

In the orientation shown in FIG. 11A, each pin in the set of pins 172preferably has a circular cross section and round ends, configured tofacilitate sliding within a pin housing 175, sliding over a cam card 177surface, and occlusion of a fluidic pathway 220. Alternatively, each pinmay comprise any appropriate cross-sectional geometry (e.g.,rectangular) and/or end shape (e.g., flat or pointed) to facilitateocclusion of a fluidic pathway 220. Preferably, the surface of each pinin the set of pins 172 is composed of a low-friction material tofacilitate sliding motions (i.e., over a cam card 177 or within a pinhousing 175); however, each pin may alternatively be coated with alubricant configured to facilitate sliding motions.

The pin housing 175 functions to constrain and guide the motion of eachpin in the set of pins 172, as the cam card 177 slides under the set ofpins 172. Preferably, the pin housing 175 comprises a set of pin housingchannels 169 configured to surround at least one pin in the set of pins172. In one variation, each pin in the set of pins 172 is surrounded byan individual channel of the set of pin housing channels 169; however,in another variation a channel of the set of pin housing channels 169may be configured to surround multiple pins in the set of pins 172. Inan example shown in FIGS. 7D-7E and 11A, the pin housing is locatedunder the cartridge platform 141, such that the set of pin housingchannels 169 is aligned with the set of valve actuation accommodatingslots 145, to provide access, by the set of pins 172, to a microfluidiccartridge 210 aligned on the cartridge platform 141. In the example, thepin housing 175 thus constrains the set of pins 172, such that each pincan only move linearly in a vertical direction. Each pin housing channelpreferably has a constricted region 168 configured to limit the motionof a pin within a pin channel; however, each pin housing channel mayalternatively not include a constricted region. Preferably, surfaces ofthe pin housing 175 contacting the set of pins 172 are composed of a lowfriction material to facilitate sliding of a pin within a pin housingchannel; however, surfaces of the pin housing 175 contacting the set ofpins 172 may alternatively be coated with a lubricant configured tofacilitate sliding motions. Other variations of the pin housing 175 andthe set of pins 172 may include no additional provisions to facilitatesliding of a pin within a pin housing channel.

In some embodiments of the molecular diagnostic module 130, the valveactuation subsystem 170 can be configured in any other suitable mannerto facilitate actuation of a set of pins 172 to occlude a microfluidiccartridge 210 at a set of occlusion positions 226. In one embodiment,the valve actuation subsystem 170 can be an embodiment of the valveactuation subsystem described in U.S. application Ser. No. 14/229,396,entitled “System and Method for Processing Biological Samples” and filedon 28 Mar. 2014, which is incorporated herein in its entirety by thisreference.

1.2.4 Molecular Diagnostic Module—Optical Subsystem

As shown in FIGS. 12A-12D, the optical subsystem 180 of the moleculardiagnostic module 130 comprises a set of light emitting diodes (LEDs)181, a set of excitation filters 182 configured to transmit light fromthe set of LEDs 181, a set of dichroic mirrors 183 configured to reflectlight from the set of excitation filters 182 toward a set of apertures185 configured to transmit light toward a set of nucleic acid samples, aset of emission filters 186 configured to receive and transmit lightemitted by the set of nucleic acid samples, and a set of photodetectors187 configured to facilitate analysis of light received through the setof emission filters 186. The optical subsystem 180 may further comprisea set of lenses 184 configured to focus light onto the set of nucleicacid samples. The optical subsystem 180 thus functions to transmit lightat excitation wavelengths toward a set of nucleic acid samples and toreceive light at emission wavelengths from a set of nucleic acidsamples. Preferably, the optical subsystem 180 is coupled to an opticalsubsystem actuator 188 configured to laterally displace and align theoptical subsystem 180 relative to a set of nucleic acid samples at a setof detection chambers 213 of the microfluidic cartridge 210, and isfurther coupled to a linear actuator 146 of the cartridge receivingmodule 140 to position the optical subsystem 180 closer to the set ofnucleic acid samples. Alternatively, the optical subsystem 180 may notbe coupled to a linear actuator 146 of the cartridge receiving module140, and may only be configured to translate laterally in one direction.In a specific example, the optical subsystem 180 comprises a set of 12apertures, a set of 12 lenses, a set of 12 dichroic mirrors, a set of 12excitation filters, a set of 12 LEDs, a set of 12 emission filters, anda set of 12 photodetectors. In a variation of the specific example, eachset of 12 optical subsystem 180 elements is configured as two sets of 6apertures, two sets of 6 lenses, two sets of 6 dichroic mirrors, twosets of 6 excitation filters (e.g., 6 excitation filters spanningdifferent wavelengths of light), two sets of 6 LEDs, two sets of 6emission filters (e.g., 6 emission filters spanning differentwavelengths of light, and two sets of 6 photodetectors, such that theoptical subsystem 180 includes two sets of identical optical subsystemunits, each comprising 6 of each element. The optical subsystem 180 can,however, comprise any suitable number of identical units, in order toincrease throughput in the system 100 by decreasing the amount of timeit takes to analyze multiple biological samples. For instance, multipleidentical units can enable analyses to be performed using a unit of theoptical subsystem 180, at a desired point during sample processing(e.g., a point at which a biological sample in a microfluidic cartridgeis being heated). In variations of the optical subsystem 180 comprisingmultiple units, each unit can be configured to move independently of theother units, or can additionally or alternatively be configured to movewith the other units in a non-independent manner. For instance, oneoptical subsystem unit can be configured to move along a first axis(e.g., a horizontal axis) independently of the other optical subsystemunit(s), but can be configured to move along a second axis (e.g., avertical axis) non-independently of the other optical subsystem unit(s).

In the specific examples, as shown in FIG. 7A-7E, the optical subsystem180 is located within the molecular diagnostic module 130 and coupled tothe linear actuator 146 of the cartridge receiving module 140, suchthat, in the extended configuration 146 b of the linear actuator 146,the optical subsystem 180 can be positioned closer to a microfluidiccartridge 210 aligned within the molecular diagnostic module. Converselyin the specific example, the optical subsystem 180 is positioned awayfrom the microfluidic cartridge 210 in the retracted configuration 146 aof the linear actuator 146. In the specific example, the opticalsubsystem 180 is further coupled to an optical subsystem actuator 188configured to laterally displace the optical subsystem 180 relative tothe microfluidic cartridge 210, such that the optical subsystem 180 canbe aligned with a set of detection chambers 213 of the microfluidiccartridge 210.

Preferably, the set of LEDs 181 are not all identical but rather chosento efficiently produce a certain band of wavelengths of light, such thatlight from the set of LEDs 181 can be filtered to appropriate narrowwavelengths for analysis of nucleic acid samples. Alternatively, allLEDs in the set of LEDs 181 may be identical, and produce white lightcomprising all wavelengths of visible light that is filtered to producethe desired wavelength, in which case the LEDs may be stationary.Preferably, the set of LEDs 181 includes phosphor-based LEDs, but theset of LEDs 181 may alternatively include any LEDs configured to providelight of the desired range of wavelengths. The LEDs of the set of LEDs181 are preferably configured to emit light of wavelengths correspondingto at least one of the set of excitation filters 182, the set ofdichroic mirrors 183, and the set of emission filters 186.

The set of excitation filters 182 is configured to align with the set ofLEDs 181 in the optical subsystem 180, and functions to transmit lightat excitation wavelengths toward the set of dichroic mirrors 183 of theoptical subsystem 180. Preferably, the set of excitation filters 182 arenot identical excitation filters, but rather chosen to transmit thedifferent desired ranges of excitation wavelengths. Alternatively, allexcitation filters of the set of excitation filters 182 are identical,and configured to transmit light having a fixed range of excitationwavelengths. In one variation, the set of excitation filters 182includes band pass filters, configured to transmit light between twobounding wavelengths, in another variation, the set of excitationfilters 182 includes short pass filters configured to transmit lightbelow a certain wavelength, and in yet another variation, the set ofexcitation filters 182 includes long pass filters configured to transmitlight above a certain wavelength. Preferably, the set of excitationfilters 182 is interchangeable, such that individual excitation filtersmay be interchanged to provide different excitation wavelengths oflight; however, the set of excitation filters 182 may alternatively befixed, such that the optical subsystem 180 is only configured totransmit a fixed range of excitation wavelengths.

The set of dichroic mirrors 183 is configured to align with the set ofexcitation filters 182, and functions to receive and reflect light fromthe set of excitation filters 182 toward the detection chamber, suchthat light having a range of excitation wavelengths may be focused,through a set of apertures, onto a set of nucleic acid samples. The setof dichroic mirrors 183 also functions to receive and transmit lightfrom a set of emission filters 186 toward a set of photodetectors 187,which is described in more detail below. All dichroic mirrors in the setof dichroic mirrors 183 are preferably identical in orientation relativeto the set of excitation filters 182 and the set of emission filters186, and configured to reflect and transmit the appropriate wavelengthsof light for the given LED. Alternatively, the set of dichroic mirrors183 may include identical dichroic mirrors, with regard to orientation,light transmission, and light reflection. In a specific example, in theorientation shown in FIG. 12A, the set of excitation filters 182 isoriented perpendicular to the set of emission filters 186, with the setof dichroic mirrors 183 bisecting an angle between two planes formed bythe faces of the set of excitation filters 182 and the set of emissionfilters 186. In the specific example, light from the set of excitationfilters is thus substantially reflected at a 90° angle toward the set ofapertures 185, and light from the set of emission filters 186 passes ina substantially straight direction through the set of dichroic mirrors183 toward the set of photodetectors 187. Other variations of the set ofdichroic mirrors 183 may include any configuration of dichroic mirrors,excitation filters, and/or emission filters that enable transmission oflight of excitation wavelengths toward a set of nucleic acid samples,and transmission of light from the set of nucleic acid samples toward aset of photodetectors 187.

In one embodiment, the optical subsystem may further include a set oflenses 184 configured to align with the set of dichroic mirrors 183,which functions to focus light, from the set of excitation filters 182and reflected off of the set of dichroic mirrors 183, onto a set ofnucleic acid samples configured to emit light in response to the lightfrom the set of excitation filters 182. All lenses in the set of lenses184 are preferably identical in orientation relative to the set ofdichroic mirrors and in dimension; however, the set of lenses 184 mayalternatively comprise non-identical lenses, such that light passingthrough different lenses of the set of lenses 184 is focused differentlyon different nucleic acid samples. In a specific example, in theorientation shown in FIG. 12A, the faces of the set of lenses 184 areoriented perpendicular to the faces of the set of excitation filters182, to account for light reflection by the set of dichroic mirrors 183at a 90° angle. In the specific example, the set of lenses also includesidentical ¼″ high numerical aperture lenses. In other variations, theset of lenses 184 may be oriented in any appropriate configuration forfocusing light from the set of dichroic mirrors 183 onto a set ofnucleic acid samples, and may include lenses of any appropriatespecification (i.e., numerical aperture).

The set of apertures 185 is located on an aperture substrate 189 andconfigured to align with the set of lenses 184, and functions to allowfocused light from the set of lenses 184 to pass through to the set ofnucleic acid samples. The aperture substrate 189 is preferably coupledto the linear actuator 146 of the cartridge receiving module 140, whichallows the optical subsystem 180 to linearly translate and be positionednear and away from a microfluidic cartridge 210 aligned within themolecular diagnostic module 130. Alternatively, the aperture substrate189 may not be coupled to the linear actuator 146 of the cartridgereceiving module 140. Preferably, all apertures 185 in the set ofapertures 185 are identical, and configured to allow identical lightprofiles to be focused, through the set of lenses 184, onto a set ofnucleic acid samples. Alternatively, the set of apertures 185 may notinclude identical apertures. In one variation, each aperture in the setof apertures 185 may be individually adjustable, in order to provideindividually modifiable aperture dimensions (e.g., width, length, ordiameter) to affect light exposure. In an alternative variation, eachaperture in the set of apertures 185 is fixed. Other variations mayinclude interchangeable aperture substrates 189, such that features ofthe set of apertures (e.g., aperture dimensions, number of apertures)may be adjusted by interchanging aperture substrates 189.

The set of emission filters 186 is configured to align with the set ofdichroic mirrors, and functions to transmit emission wavelengths oflight from the set of nucleic acid samples, and to filter out excitationwavelengths of light. Preferably, each emission filter of the set ofemission filters 186 are configured to transmit light having a fixedrange of emission wavelengths, while blocking light of excitationwavelengths. Alternatively, the set of emission filters 186 may compriseidentical emission filters, such that individual emission filters of theset of emission filters 186 are configured to transmit the same rangesof emission wavelengths. In one variation, the set of emission filters186 includes band pass filters, configured to transmit light between twobounding wavelengths, in another variation, the set of emission filters186 includes short pass filters configured to transmit light below acertain wavelength, and in yet another variation, the set of emissionfilters 186 includes long pass filters configured to transmit lightabove a certain wavelength. Preferably, the set of emission filters 186is interchangeable, such that individual emission filters may beinterchanged to transmit and/or block different wavelengths of light;however, the set of emission filters 186 may alternatively be fixed,such that the optical subsystem 180 is only configured to transmit afixed range of emission wavelengths.

The set of photodetectors 187 is configured to align with the set ofemission filters 186, and functions to receive light from the setemission filters to facilitate analysis of the set of nucleic acidsamples. All photodetectors in the set of photodetectors 187 arepreferably identical; however, the set of photodetectors 187 mayalternatively include non-identical photodetectors. Preferably, the setof photodetectors 187 includes photodiodes comprising a photoelectricmaterial configured to convert electromagnetic energy into electricalsignals; however, the set of photodetectors 187 may alternativelycomprise any appropriate photodetectors for facilitating analysis ofbiological samples, as is known by those skilled in the art.

The optical subsystem actuator 188 is coupled to the optical subsystem180, and functions to laterally translate the optical subsystem 180relative to a set of nucleic acid samples being analyzed. Preferably,the optical subsystem actuator 188 is a linear actuator configured totranslate the optical subsystem 180 in one dimension; however, theoptical subsystem actuator 188 may alternatively be an actuatorconfigured to translate the optical subsystem 180 in more than onedimension. In a specific example, as shown in FIGS. 7A-7D and 12D, theoptical subsystem actuator 188 is configured to translate the opticalsubsystem 180 laterally in a horizontal plane, to align the opticalsubsystem 180 with a set of detection chambers 213 of a microfluidiccartridge 210 within the molecular diagnostic module 130. In anotherexample, the optical subsystem may be configured as a disc revolvingaround an axis with the LEDs and photodetectors stationary and the disccontaining the filters. In other variations, the optical subsystemactuator 188 may be configured in any appropriate manner to facilitatealignment of the optical subsystem 180 relative to a set of nucleic acidsamples being analyzed.

In some variations, wherein reflection from the set of detection chamberheaters 157 and/or any other element of the system interferes with lightemitted directly from biological samples at the microfluidic cartridge210 or light transmitted through the set of excitation filters 182, theoptical system can be configured to filter out undesired reflectedlight, by way of any one or more of: the set of excitation filters 182,the set of dichroic mirrors 183, the set of emission filters 186, andany other suitable element configured to reduce or remove interferencecaused by undesired reflected light.

The optical subsystem 180 can, however, comprise any other suitableelement(s) and/or be configured in any other suitable manner tofacilitate analysis of a set of biological samples.

1.2.5 Molecular Diagnostic Module—Alternative Embodiments and Variations

As described above, alternative embodiments of the molecular diagnosticmodule 130 and alternative variations of subsystems and elements of themolecular diagnostic module 130 may be configured to process abiological sample containing nucleic acids, isolate nucleic acids fromthe biological sample, and detect nucleic acids. An example of analternative embodiment of a molecular diagnostic module 130, as shown inFIG. 13, includes a cartridge receiving module 140′, a heating andcooling subsystem 150′, a magnet 160′, a valve actuation subsystem 170′,and an optical subsystem 180′, and functions to manipulate analternative microfluidic cartridge 210′ for processing of biologicalsamples containing nucleic acids. Other alternative embodiments of themolecular diagnostic module 130″ may be configured to receivealternative microfluidic cartridges 210″, for processing of biologicalsamples containing nucleic acids.

1.3 System—Assay Strip

As shown in FIGS. 18A and 18B, the assay strip 190 comprises an assaystrip substrate 191 comprising a set of wells 192, and typically apuncturable foil seal 195, and functions to facilitate combination of aset of nucleic acid samples with a set of molecular diagnostic reagentsfor amplification and/or detection of a nucleic acid sequence orsequences. Preferably, the entire assay strip 190 is configured to be aconsumable (i.e., disposable), such that the assay strip 190 can be usedduring multiple runs of the system 100, then the assay strip 190 isdisposed of once all of the wells 192, containing unitized reagents fora single test or group of tests, is exhausted. Alternatively, at least aportion of the assay strip 190 is configured to be reusable, such thatwells may be reloaded with reagents and reused with the system 100. Inone variation of the assay strip 190, the assay strip substrate 191 isreusable, while the puncturable foil seal 195 is disposable and replacedafter each run of the system 100. In another variation, the reusableassay strip substrate 191 does not require a puncturable foil seal 195,such that reagents specific to a certain nucleic acid sequences may bedeposited into open wells of the assay strip substrate 191 by a user.

The assay strip substrate 191 is configured such that the assay strip190 is capable of resting on a flat surface, and functions to define theset of wells 192 and to couple to the puncturable foil seal 195. Theassay strip substrate 191 is preferably configured to be received by acorresponding assay strip holder 230 configured to hold multiple assaystrips 190, but may alternatively not be configured to couple to anassay strip holder 230. The assay strip substrate 191 is preferablycomposed of a PCR-compatible polymer, such as polypropylene, that can beheat processed to couple to the puncturable foil seal 115, but canalternatively be composed of any appropriate material that can contain afluid and be bonded to the puncturable foil seal 115.

The set of wells 192 of the assay strip substrate 191 function toreceive at least one nucleic acid sample, and to facilitate combinationof the nucleic acid sample with at least one of a set of moleculardiagnostic reagents. The molecular diagnostic reagents of the set ofmolecular diagnostic reagents preferably comprise reagents configured toanalyze the set of nucleic acid volumes for markers of at least one ofgonorrhea (GC), Chlamydia (CT), herpes simplex virus (HSV), humanimmunodeficiency virus (HIV), human respiratory diseases, vaginaldiseases, hepatitis C virus (HCV), hepatitis B virus (HBV), trichonomas,group B Streptococcus (GBS), factor 2 (FII) gene, and factor five (FV)gene, but may alternatively comprise reagents used to performalternative molecular diagnostic protocols (e.g., for DNA/RNA targetsderived from one or more of bacteria, viruses, human mRNA, microRNA,etc.). Preferably, the wells 193 of the assay strip substrate 191 areeach configured to accommodate not only a nucleic acid sample, but alsoto facilitate mixing of the nucleic acid sample with at least one of aset of molecular diagnostic reagents (e.g., using a pipettor or otherapparatus). Additionally, the molecular diagnostic reagents of the setof molecular diagnostic reagents preferably comprises probes and primersto detect the sample process controls provided by the capture plate, inorder to verify process fidelity and assay accuracy. Preferably, thewells 193 are deep enough to facilitate mixing without splashing, andevenly spaced to facilitate aspiration, delivery, and/or mixing ofmultiple biological samples (e.g., with a multi-tip pipettor).Alternatively, the wells are wide and shallow to facilitate drying ofreagents in the wells to increase shelf life and larger devices formixing the nucleic acids with molecular diagnostic reagents. Each well193 of the set of wells 192 also preferably has a rounded bottom region,as shown in FIG. 18A, to facilitate complete aspiration of a fluid froma well 193; however, each well 193 may alternatively not have a roundedbottom region. Additionally, the set of wells 192 is preferably arrangedin staggered rows, which functions to facilitate access to individualwells 193 of the set of wells, to reduce one dimension of the assaystrip 190, and also to prevent cross-contamination of fluids within thewells due to dripping. Alternatively, the set of wells 192 may not bearranged in staggered rows.

The puncturable foil seal 195 functions to protect the moleculardiagnostic reagents stored in wells 112 from degradation, isolate eachwell 193 of the set of wells 192, prevent contamination of the contentsof each of the set of wells 192, and provide information identifying theassay strip 190. The puncturable foil seal 195 preferably seals eachwell 193 of the assay strip 190, and is configured to be punctured by anexternal element (e.g., by a pipette tip), such that each well is sealedprior to being punctured. In one variation, the puncturable foil seal195 also forms a seal around an element that punctures it, and inanother variation, the puncturable foil seal 195 does not form a sealaround an element that punctures it, in order to prevent airlock. Thepuncturable foil seal 195 is also preferably labeled with identifyinginformation including at least one of manufacturer information, assaystrip contents, the lot of the contents, an expiry date, and a uniqueelectronic tag (e.g., barcode or QR code) providing more information.Preferably, the puncturable foil seal 195 does not extend beyond thefootprint of the assay strip 190, but alternatively, the puncturablefoil seal 195 may be any appropriate size and/or include protrudingfeatures (e.g., tabs) that facilitate handling of the assay strip.

In one variation, the assay strip 190 may be prepackaged with a set ofmolecular diagnostic reagents, such that each well 193 in the set ofwells 192 is prepackaged with a quantity of molecular diagnosticreagents. The set of wells 192 may then be sealed by the puncturablefoil seal 195, which is configured to be punctured by an externalelement that delivers volumes of nucleic acid samples to be combinedwith the set of molecular diagnostic reagents. In another variation, theassay strip 190 may not be prepackaged with a set of moleculardiagnostic reagents, and the wells 193 of the assay strip 190 may not besealed with a puncturable foil seal 195. In yet another variation, thesystem may comprise an empty assay strip 190 without a puncturable foilseal 195, and an assay strip 190 comprising reagents and a puncturablefoil seal 195, such that a user may add specific reagents to the emptyassay strip to be used in conjunction with the assay strip comprisingreagents. In variations comprising a puncturable foil seal 195, thepuncturable foil seal 115 is configured to be punctured by at least oneexternal element, for co-delivery of nucleic acid samples and moleculardiagnostic reagents intended to be combined.

In a specific example, the assay strip 190 has an 87 mm×16 mm footprintand comprises 24 wells 113 arranged in two staggered rows, with a 9 mmcenter-to-center pitch between adjacent wells 193 within each row. Eachwell 193 of the set of wells has a capacity of 60 μL to accommodate avolume of a molecular diagnostic reagent, 20 μL of a sample fluid, andany displacement caused by a pipette tip (e.g., 100 or 300 μL pipettetip). Each well 113 of the assay strip 190 in the specific example isalso prepackaged with a quantity of molecular diagnostic reagents, andcomprises a protruding top edge (75 microns high) that is heat sealed toa puncturable foil seal. The capture plate 110 in the specific exampleis produced by injection molding, has a footprint of 127.75 mm×85.5 mm,and is composed of a PCR-compatible polypropylene based polymer with ahigh vapor barrier. In the specific embodiment, the vapor barrier isfurther increased by depositing a thin metallic layer to the outside ofthe assay strip 190.

As described earlier, the assay strip 190 may be configured to bereceived by an assay strip holder 230. The assay strip holder 230functions to receive and align multiple assay strips 190, such that amultichannel pipettor or other fluid delivery system may combinemultiple nucleic acid samples with molecular diagnostic reagents usingwells 193 of multiple assay strips 190. In one variation, the assaystrip holder 230 may be configured to contain Assay strips 190 includingreagents for substantially different molecular diagnostic assays, asshown in FIG. 17B, such that a single run of the system 100 involvesanalyzing a set of nucleic acid samples under different moleculardiagnostic assays. In another variation, the assay strip holder 230 maybe configured to contain assay strips 190 including reagents foridentical molecular diagnostic assays, such that a single run of thesystem 100 involves analyzing a set of nucleic acid samples under thesame molecular diagnostic assay. Preferably, the assay strip holder 230is composed of a material that is dishwasher safe and autoclavable,configured to hold the assay strips 190 in place during handling by afluid delivery system (e.g., pipettor), and configured such that theassay strips 190 avoid protruding over an edge of the assay strip holder230, but the assay strip holder 230 is constructed to facilitateinsertion and removal of the assay strips 190 from the assay stripholder 230.

In one variation, the assay strip holder 230 is not configured tofacilitate cooling of molecular diagnostic reagents within the assaystrips 190; however, in another variation as shown in FIG. 21A, theassay strip holder 230 may be further configured to couple to analuminum block 235 coupled to a set of Peltier units 236 configured tofacilitate cooling of molecular diagnostic reagents within the assaystrips 190. Additionally, the assay strip holder 230 may be configuredto be received and carried by an assay strip carrier 240, which, asshown in FIG. 20, functions to facilitate handling and alignment ofmultiple assay strip holders 230. In a specific example, as shown inFIG. 19, the assay strip holder 230 has dimensions of 127.76 mm×85.48mm×14.35 mm, complies with American National Standards Institute (ANSI)and Society for Laboratory Automation and Screening (SLAS) standards,and is configured to hold six 16-well assay strips for a total of 96wells 193. In another specific example, as shown in FIG. 21B, the assaystrip holder 230′ is configured to hold four assay strips 190′, eachcomprising 24 wells 193′ for a total of 96 wells per assay strip holder230′. Other combinations of the described embodiments, variations, andexamples of the assay strip 190, assay strip holder 230, and assay stripcarrier 240 may be incorporated into embodiments of the system 100 forprocessing and detecting nucleic acids.

1.4 System—Microfluidic Cartridge

The microfluidic cartridge 210 functions to receive a set of magneticbead-samples, facilitate separation of nucleic acids from the set ofmagnetic bead-samples, receive a set of nucleic acid-reagent samples,and facilitate analysis of nucleic acids from the set of nucleicacid-reagent samples. In one embodiment, the microfluidic cartridge 210comprises a top layer 211 including a set of sample port-reagent portpairs 212 and a set of detection chambers 213; an intermediate substrate214, coupled to the top layer 211 and partially separated from the toplayer 211 by a film layer 215, configured to form a waste chamber 216;an elastomeric layer 217 partially situated on the intermediatesubstrate 214; a magnet housing region 218 accessible by a magnet 160providing a magnetic field; and a set of fluidic pathways 219, eachformed by at least a portion of the top layer 211, a portion of the filmlayer 215, and a portion of the elastomeric layer 217. In theembodiment, the microfluidic cartridge 10 further comprises a bottomlayer 221 coupled to the intermediate substrate 214 and configured toseal the waste chamber 216. Furthermore, in the embodiment, the toplayer 211 of the microfluidic cartridge 210 further comprises a sharedfluid port 222, a vent region 223, and a heating region 224, such thateach fluidic pathway 220 in the set of fluidic pathways 219 isfluidically coupled to a sample port-reagent port pair 224, the sharedfluid port 222, the waste chamber 216, and a detection chamber 225,comprises a turnabout portion 226 configured to pass through the heatingregion 224 and the magnetic field, and is configured to pass through thevent region 223 upstream of the detection chamber 225. Each fluidicpathway 220 thus functions to receive and facilitate processing of asample fluid containing nucleic acids as it passes through differentportions of the fluidic pathway 220.

The microfluidic cartridge 210 is preferably configured to be receivedand manipulated by the molecular diagnostic module 130, such that thecartridge receiving module 140 of the molecular diagnostic module 130receives and aligns the microfluidic cartridge 210 within the moleculardiagnostic module 130, the heating and cooling subsystem 150 of themolecular diagnostic module 130 is configured to transfer heat to theheating region 224 of the microfluidic cartridge 210, and the magnet 160of the molecular diagnostic module 130 is configured to be received bythe magnet housing region 218 of the microfluidic cartridge 210 toprovide a magnetic field for separation of nucleic acids. Additionally,the shared fluid port 222 of the microfluidic cartridge 210 isconfigured to couple to a nozzle 149 coupled to the linear actuator 146of the cartridge receiving module 140, such that the liquid handlingsystem 250 can deliver fluids and gases through the shared fluid port222. The elastomeric layer 217 of the microfluidic cartridge 210 is alsopreferably configured to be occluded at a set of occlusion positions 226by the valve actuation subsystem 170 of the molecular diagnostic module,in order to occlude portions of a fluidic pathway 220 of themicrofluidic cartridge 210 for processing of a set of biologicalsamples. The optical subsystem 180 of the molecular diagnostic module130 is further configured to align with the set of detection chambers213 of the microfluidic cartridge 210, to facilitate analysis of a setof nucleic acid samples. The microfluidic cartridge 210 is preferablythe microfluidic cartridge 210 described in U.S. application Ser. No.13/765,996, which is incorporated in its entirety by this reference, butmay alternatively be any appropriate cartridge or substrate configuredto receive and process a set of samples containing nucleic acids.

1.5 System—Fluid Handling System and Filter

The liquid handling system 250 of the system 100 includes a liquidhandling arm 255 and a syringe pump 265, as shown in FIGS. 14A-14C andfunctions to deliver biological samples, reagents, and gases to elementsof the system 100. As described in Section 1, an embodiment of theliquid handling system 250 is configured to aspirate a set of biologicalsamples containing nucleic acids (i.e., impure nucleic acid samples),dispense the set of biological samples into a capture plate 110 to belysed and combined with magnetic beads by a capture plate module 120,aspirate the set of biological samples combined with magnetic beads(i.e., set of magnetic bead-samples) from the capture plate 110, anddispense the set of magnetic bead-samples into microfluidic cartridge210 located in a molecular diagnostic module 130. The embodiment of theliquid handling system 100 is further configured to facilitateseparation of a set of nucleic acids from the magnetic bead-samples, bydispensing a wash solution, a release solution, and/or air into themolecular diagnostic module 130, by the nozzle 149 coupled to the linearactuator 146, at appropriate stages, aspirate the set of nucleic acidsfrom the molecular diagnostic module 130, combine the set of nucleicacids with a set of molecular diagnostic reagents using an assay strip190, and dispense the set of nucleic acids combined with the set ofmolecular diagnostic reagents (i.e., set of nucleic acid-reagentmixtures) into the molecular diagnostic module 130 for furtherprocessing and analysis. Other embodiments of the liquid handling system250 may be configured to perform alternative molecular diagnostic assayprotocols and/or dispense and aspirate alternative fluids into and fromother elements supporting a molecular diagnostic protocol.

The liquid handling arm 255 comprises a gantry 256 and a multichannelliquid handling head 257, and functions to travel to different elementsof the system 100 for fluid delivery and aspiration. The liquid handlingarm 255 is preferably automated and configured to move, aspirate, anddeliver fluids automatically, but may alternatively be a semi-automatedliquid handling arm 255 configured to perform at least one of moving,aspirating, and delivering automatically, while another entity, such asa user, performs the other functions.

The gantry 256 is coupled to the multichannel liquid handling head 257,and functions to transport the multichannel liquid handling head 257 todifferent elements of the system 100 for fluid delivery and aspiration.Preferably, the gantry 256 is automated and configured to translate themultichannel liquid handling head 257 within at least two dimensions,and provides X-Y positional accuracy of at least 0.5 mm. Additionally,in the orientation shown in FIG. 14B, the gantry is preferably situatedabove the molecular diagnostic module 130, such that the gantry 256 cantranslate within at least two dimensions without interfering with otherelements of the system 100. Alternatively, the gantry 256 may be anyappropriate gantry 256 to facilitate movement of an end effector withinat least two dimensions, as is readily known by those skilled in theart.

The multichannel liquid handling head 257 functions to aspirate fluidsfrom and deliver fluids to different elements of the system 100.Preferably, the multichannel liquid handling head 257 is a multichannelpipette head; however, the multichannel liquid handling head 257 mayalternatively be any appropriate multichannel liquid handling headconfigured to deliver fluids and/or gases. Preferably, the multichannelliquid handling head 257 comprises at least eight independent channels258, but may alternatively comprise any number of channels 258configured to aspirate and deliver fluids. The channel-to-channel pitchis preferably variable, and in a specific example ranges between 9 mmand 36 mm; however, the channel-to-channel pitch may alternatively befixed, as shown in FIG. 15. The multichannel liquid handling head 257also preferably provides independent z-axis control (in the orientationshown in FIG. 14B), such that, in combination with the gantry 256. Themultichannel liquid handling head 257 is preferably configured to coupleto both large (e.g., 1 mL) and small (e.g., between 100 and 300 μL)pipette tips, and in a specific example, has a precision of at least 6%using small disposable pipette tips and a precision of at least 2% usinglarge disposable pipette tips when dispensing essentially the entire tipvolume. Alternatively, the multichannel liquid handling head 257 may beconfigured to couple to any object configured to facilitate aspirationand delivery of fluids. Preferably, the multichannel liquid handlinghead 257 provides independent control of the channels 258, with regardto volumes of fluid aspirated or delivered, fluid dispensing rates,and/or engaging and disengaging pipette tips. Alternatively, themultichannel liquid handling head 257 may not provide independentcontrol of the channels 258, such that all channels 258 of themultichannel liquid handling head 257 are configured to performidentical functions simultaneously. Preferably, the multichannel liquidhandling head 257 is configured to aspirate and deliver both liquids andgases, but alternatively, the multichannel liquid handling head 257 maybe configured to only aspirate and deliver liquids. Preferably, themultichannel liquid handling head 257 provides at least one of liquidlevel detection, clot detection, and pipette tip engaging/disengagingdetection for each of the channels 258; however, the multichannel liquidhandling head 257 may alternatively not provide liquid level detection,clot detection, and pipette tip engaging/disengaging detection for eachof the channels 258.

In one embodiment, the multichannel liquid handling head 257 isconfigured to couple to at least one filter 260, which functions topre-filter liquids being aspirated and/or dispensed by the liquidhandling arm 255, and is preferably a custom filter 260 configured tocouple to a pipette tip, but may alternatively be any appropriate filterconfigured to couple to the liquid handling arm 255 and filter liquidsbeing aspirated and/or dispensed by the liquid handling arm 255.

An embodiment of a custom filter 260, as shown in FIG. 22, comprises afirst end 261 configured to couple to a pipette tip, a pointed secondend 262, a void 263 coupled to the first end 261 and the pointed secondend 262, and a filter membrane 264 subdividing the void 263. The firstend 261, as shown in FIG. 22, preferably comprises a tapered channelconfigured to provide a friction fit with a pipette tip; however, thefirst end may alternatively not comprise a tapered channel and may beconfigured to couple to a pipette tip using any appropriate means. Thepointed second end 262 is preferably sharp and configured to pierce anobject, such as a foil seal; additionally, the pointed second end 262 ispreferably at least as long as required to dispense into a well 113 ofthe capture plate 110. The void 263 preferably defines a conical regiondefined by the filter membrane 264, wherein the conical region isconfigured to divert a fluid within the filter 260 toward the pointedsecond end 262; however, the void 263 may not include a conical region.The filter membrane 264 functions to filter a fluid aspirated by themultichannel liquid handling head 257, and is configured to subdividethe void 263 to define a conical region; however, the filter membrane264 may alternatively not define a conical region of the void 263. Inone embodiment, in the orientation shown in FIG. 22, the region of thevoid 263 below the filter membrane 264 may have a volumetric capacity ofbetween 200 ul and 1 mL; however, the region of the void 263 below thefilter membrane may alternatively have any appropriate volumetriccapacity.

A set of filters 260 may further be configured to be received anddelivered by a filter holder 269, as, shown in FIG. 23. A specificembodiment of a filter holder 269 comprises a set of 24 tapered holeswith an 18 mm center-to-center pitch, arranged in six rows of fourholes. The specific embodiment of the filter holder 269 is alsocompliant with ANSI and SLAS standards, has dimensions of127.75×85.5×14.35 mm, and is stackable with other specific embodimentsof the custom filter holder 269. Alternatively, the filter holder 269may be any appropriate filter holder 269 configured to receive anddeliver a set of filters 260, as is readily known by those skilled inthe art.

1.5.1 Fluid Handling System—Syringe Pump

The syringe pump 265 of the liquid handling system 250 is coupled to awash solution source 266, a release solution source 267, a source of air268, and flexible tubing 291, and functions to deliver a wash solution,a release solution, and air through a valve to the molecular diagnosticmodule 130 to facilitate isolation and purification of nucleic acidsfrom a set of magnetic bead-samples. The flexible tubing 291 ispreferably coupled at a first end to the syringe pump, and at a secondend to a nozzle 149 coupled to the linear actuator 146 of the moleculardiagnostic module 130, as shown in FIG. 14C. As stated earlier, anextended configuration 146 b of the linear actuator 146 is configured tocouple the nozzle 149 to a fluid port 222 of a microfluidic cartridge210 within the molecular diagnostic module 130, such that the washsolution, release solution, and air can be delivered to the microfluidiccartridge 210 at appropriate stages. A specific embodiment of thesyringe pump 265 comprises a 4-way valve, is able to pump 20-5000 μL offluids or air through the 4-way valve at flow rates from 50-500 μL/min,can couple to syringes with between 1 mL and 10 mL capacities, and has aprecision of at least 5% with regard to fluid or air delivery.Alternatively, the syringe pump 265 may be any appropriate syringe pump265 or fluid delivery apparatus configured to deliver a wash solution, arelease solution, and air to the molecular diagnostic module 130, as isreadily known by those skilled in the art.

1.6 System—Additional Elements

The system 100 may further comprise a tag reader 271, which functions toread barcodes, QR codes and/or any other identifying tags of the system100. Preferably, the tag reader 271 is coupled to the liquid handlingsystem 250, such that the tag reader 271 is configured to read tags onpuncturable foil seals 115, 195 or tags located on any element of thesystem 100 accessible by the liquid handling system 250; however, thetag reader 271 may alternatively not be coupled to the liquid handlingsystem 250. In one alternative embodiment of the system 100, the tagreader 271 may be a standalone unit that is configured to be manipulatedby a user to scan tags or labels located on elements of the system 100.

The system 100 may also further comprise a controller 272 coupled to atleast one of the capture plate module 120, the molecular diagnosticmodule 130, the liquid handling system 250, and the tag reader 271, andfunctions to facilitate automation of the system 100. In a variationwherein the controller 272 is coupled to the capture plate module 120,the controller 272 preferably functions to automate heating of a captureplate 110, which facilitates lysing of biological samples within thecapture plate 110 and binding of nucleic acids within the capture plate110 to magnetic beads 119 of the capture plate 110. In a variationwherein the controller 272 is coupled to the molecular diagnostic module130, the controller 272 preferably functions to automate reception of amicrofluidic cartridge, heating of biological samples within themolecular diagnostic module 130 and the detection chambers 213,occlusion of fluidic pathways 220 by the valve actuation subsystem 170,and analysis of a set of nucleic acid-reagent mixtures by the opticalsubsystem 180. In a variation wherein the controller 272 is coupled tothe liquid handling system 250, the controller 272 preferably functionsto automate aspiration, transfer, and delivery of fluids and/or gases todifferent elements of the system 100. In a variation wherein thecontroller 272 is coupled to the tag reader 271, the controllerpreferably functions to automate reading of tags by the tag reader 271,and may further function to facilitate transfer of information from thetags to a processor 273. Other variations of a controller may functionautomate handling, transfer, and/or storage of other elements of thesystem 100, such as capture plates 110, assay strips 190, assay stripholders 230, assay strip carriers 240, filters 200, filter holders 205,and/or microfluidic cartridges 210, using a robotic arm (or otherrobotic module or robotic component) or gantry similar to that used inthe liquid handling system 250. Alternative combinations of the abovevariations may involve a single controller 272, or multiple controllersconfigured to perform all or a subset of the functions described above.

The system 100 may also further comprise a processor 273, whichfunctions to receive and process information from a tag reader 271, andalso to receive and process data received from the optical subsystem 180of the molecular diagnostic module 130. Preferably, the processor 273 iscoupled to a user interface 274, which functions to display processedand/or unprocessed data produced by the system 100, settings of thesystem 100, information obtained from a tag reader 271, or any otherappropriate information. Alternatively, the processor 273 is not coupledto a user interface 274, but comprises a connection 275 configured tofacilitate transfer of processed and/or unprocessed data produced by thesystem 100, settings of the system 100, information obtained from a tagreader 271, or any other appropriate information to a device external tothe system 100.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made the described embodiments of the system 100 withoutdeparting from the scope of the system 100.

2. Method for Processing and Detecting Nucleic Acids

An embodiment of a method 400 for processing and detecting nucleic acidsfrom a set of biological samples comprises: combining each biologicalsample of the set of biological samples with a quantity of magneticbeads to produce a set of magnetic bead-sample mixtures S410; heatingthe set of magnetic bead-sample mixtures to produce a set of nucleicacid-magnetic bead samples S420; transferring each nucleic acid-magneticbead sample of the set of nucleic acid-magnetic bead samples to acorresponding fluidic pathway of a set of fluidic pathways S430;producing a set of nucleic acid volumes from the set of nucleicacid-magnetic bead samples S440; combining each nucleic acid volume ofthe set of nucleic acid volumes with a molecular diagnostic reagent of aset of molecular diagnostic reagents to produce a set of nucleicacid-reagent mixtures S450; transferring each of the set of nucleicacid-reagent mixtures, through the corresponding fluidic pathway of theset of fluidic pathways, to a detection chamber of a set of detectionchambers S460; and receiving light from the set of nucleic acid-reagentmixtures S470. The method 400 may further comprise generating a set ofdata based on light received form the set of nucleic acid-reagentmixtures S480. The method 400 functions to isolate and extract a set ofnucleic acid volumes from a biological sample, and to facilitateanalysis of the nucleic acid volumes according to at least one moleculardiagnostic protocol. In relation to sample processing, the processor ofthe system can include non-transitory computer-readable medium withinstructions for executing a reagent receiving mode with movement of theaspiration and delivery head of the fluid handling system into fluidcontact with at least one storage container, and executing a reagentdelivery mode with movement of the aspiration and delivery head of thefluid handling system into contact with a port of the cartridge, asdescribed in more detail below.

Step S410 recites combining each biological sample of the set ofbiological samples with a quantity of magnetic beads to produce a set ofmagnetic bead-sample mixtures, and functions to prepare a set ofbiological samples to be lysed and combined with magnetic beads. Foreach biological sample, Step S410 preferably comprises aspirating aportion of the volume of the biological sample from a sample container(possibly containing an aqueous solution prior to addition of biologicalsample), and transferring the portion of the biological sample to a wellcontaining a set of magnetic beads. Alternatively, for each biologicalsample, Step S410 may comprise aspirating the entire volume of thebiological sample from a sample container, and transferring the volumeof the biological sample to be combined with a set of magnetic beads.Preferably, all biological samples in the set of biological samples areaspirated and combined with the magnetic beads in the wellssimultaneously using a multichannel fluid delivery system; however, allbiological samples in the set of biological samples may alternatively beaspirated and combined with a set of magnetic beads non-simultaneously.The magnetic beads are preferably polymer beads, precoupled with aligand for binding to a nucleic acid, and comprising asuperparagmagnetic component. Additionally, the magnetic beads may betreated to be positively charged. However, the magnetic beads mayalternatively be any appropriate magnetic beads configured to facilitatebiomagnetic separation.

In addition to combination with magnetic beads, Step 410 may furtherinclude combining each biological sample of the set of biologicalsamples with a lysing enzyme (e.g. proteinase K), and a sample processcontrol comprising two or more nucleic acid sequences (i.e., one for DNAand one for RNA) to be included with each sample. This allows biologicalsamples to effectively lysed, which releases waste components into awash solution, and allows nucleic acids to bind to magnetic beads. Thisadditionally allows the sample process control to be later detected, asa check to verify the accuracy of a molecular diagnostic assay beingperformed.

In a first variation of Step S410 for one biological sample, as shown inFIG. 16A, a volume of the biological sample is aspirated and combinedwith a set of magnetic beads. In the first variation of Step S410, a setof different biological samples may thus be aspirated simultaneously,and each biological sample may be transferred to an individual well tobe combined with a set of magnetic beads to produce a set of magneticbead-sample mixtures. In the first variation of Step S410, all magneticbead-sample mixtures in the set of magnetic bead-sample mixtures aresubstantially non-identical in composition. In a second variation ofStep S410, as shown in FIG. 16B, a volume of a stock biological sampleis aspirated, and portions of the volume of the stock biological sampleare transferred to multiple wells to be combined with multiple sets ofmagnetic beads to produce a set of magnetic bead-sample mixtures. In thesecond variation of Step S410, all magnetic bead-sample mixtures in theset of magnetic bead-sample mixtures are substantially identical incomposition. Other variations of Step S410 may additionally oralternatively comprise filtering at least one biological sample of theset of biological samples S415 prior to combining each biological sampleof the set of biological samples with a quantity of magnetic beads.

In a specific example of Step S410, a multichannel liquid handlingsystem aspirates approximately 1 mL of each of a set of biologicalsamples in aqueous buffer using a set of 1 mL pipette tips, couples eachof the pipette tips to a custom 13 mm diameter filter, punctures a foilseal 115 of a capture plate at a set of wells, wherein each well of theset of wells contains a set of magnetic beads, and dispenses eachaspirated volume of a biological sample into a well of the capture platecontaining a set of magnetic beads, and disposes of the tip/filtercombination. In the specific example of Step S410, the multichannelliquid handling system then picks up new disposable tips and aspiratesand dispenses the contents of each well of the set of wells of thecapture plate at least three times to mix the contents, and thendisposes of the set of pipette tips and filters.

Additionally or alternatively, the method 400 can include combining eachbiological sample of the set of biological samples with buffer solutionS405 prior to combining each biological sample of the set of biologicalsamples with a quantity of magnetic beads, which functions to furtherdecrease sample preparation burden on an entity processing and/oranalyzing the set of biological samples. Combining each biologicalsample of the set of biological samples with buffer solution can beperformed using a buffer plate comprising a set of wells, each wellcontaining buffer solution, wherein the buffer plate is included in anembodiment of the system 100 described above; however, Block S405 canadditionally or alternatively be implemented using any other suitableportion of the system 100 described above. In one variation, a liquidhandling system can be automatically configured to aspirate the set ofbiological samples and mix the biological samples with the buffersolution within the buffer plate, by dispensing the biological samplesinto the buffer plate and aspirating and delivering the samples combinedwith buffer one or more times. In another variation, combination of theset of biological samples with buffer solution can be implemented by theliquid handling system away from the buffer plate, for instance, byaspirating the set of biological samples, aspirating the buffersolution, and then mixing the biological samples with the buffersolution within the liquid handling system. In other variations, thebiological samples can be combined with buffer solution in any othersuitable manner (e.g., altogether with mixing the biological sample withmagnetic beads). In relation to the specific example of Block S410described above, the multichannel liquid handling system can beconfigured to aspirate each of the set of biological samples with a setof pipette tips, dispense each of the set of biological samples into acorresponding well of a set of wells containing buffer solution, mixeach of the set of biological samples with the buffer solution using theset of pipette tips, while preventing cross contamination across the setof samples by way of the set of pipette tips, couple the set of pipettetips to a custom filter, and push the biological samples mixed withbuffer solution into wells of a capture plate, each well of the captureplate containing a set of magnetic beads. As such, mixing of biologicalsamples with buffer can be performed in an automated manner using anembodiment of the system 100 described above, prior to mixing thebiological samples with magnetic beads in Block S410.

Step S420 recites heating the set of magnetic bead-sample mixtures toproduce a set of nucleic acid-magnetic bead samples, and functions toincubate the set of magnetic bead-sample mixtures in order to lysebiological matter, and release nucleic acids to be bound to magneticbeads. Preferably, Step S420 comprises heating a capture platecontaining the set of magnetic bead-sample mixtures for a specifiedamount of time at a specified temperature, and may additionally includecooling the set of magnetic bead-sample mixtures. In a specific example,Step S420 comprises heating a capture plate containing the set ofmagnetic bead-sample mixtures using a capture plate module, wherein thecapture plate module is configured to cradle and controllably heat wellscontaining the set of magnetic bead-sample mixtures. Step S420 mayalternatively comprise incubating the set of magnetic bead-samplemixtures using any appropriate method and/or system as is known by thoseskilled in the art. Finally, Step S420 may be omitted in embodiments ofthe method 400 involving samples that do not require heating.

Step S430 recites transferring each nucleic acid-magnetic bead sample ofthe set of nucleic acid-magnetic bead samples to a corresponding fluidicpathway of a set of fluidic pathways, and functions to isolate each ofthe set of nucleic acid-magnetic bead samples within separate pathwaysfor further processing. Preferably, all nucleic acid-magnetic beadsamples in the set of nucleic acid-magnetic bead samples are transferredsimultaneously to the set of fluidic pathways, but alternatively, eachnucleic acid-magnetic bead sample in the set of magnetic bead-samplesmay be transferred to a corresponding fluidic pathway independently ofthe other nucleic acid-magnetic bead samples. In addition, preferablythe entire volume, or substantially all of the volume, of the nucleicacid-magnetic bead sample is transferred to the set of fluidic pathways,without magnetically isolating magnetic beads and removing supernatantfluids prior to transferring each nucleic acid-magnetic bead sample ofthe set of nucleic acid-magnetic bead samples to a corresponding fluidicpathway of a set of fluidic pathways.

Step S430 may further comprise occluding at least one fluidic pathway ofthe set of fluidic pathways at a subset of a set of occlusion positionsS432, which functions to define at least one truncated fluidic pathway.Preferably, Step S432 comprises defining at least one truncated fluidicpathway passing through at least one of a heating region and a magneticfield; however, Step S432 may alternatively not comprise defining atruncated fluidic pathway passing through at least one of a heatingregion and a magnetic field.

In a specific example of Step S430, the multichannel liquid handlingsubsystem of Step S410 transfers a set of nucleic acid-magnetic beadsamples to a set of fluidic pathways of a microfluidic cartridge alignedwithin a molecular diagnostic module, wherein the microfluidic cartridgecomprises an elastomeric layer in contact with the set of fluidicpathways. Manipulation of the elastomeric layer at a subset of a set ofocclusion positions by a valve actuation subsystem of the moleculardiagnostic module defines a set of truncated fluidic pathways crossing aheating region and a magnetic field, such that each nucleicacid-magnetic bead sample in the set of nucleic acid-magnetic beadsamples is isolated within a truncated fluidic pathway of the set oftruncated fluidic pathways.

Step S440 recites producing a set of nucleic acid volumes from the setof nucleic acid-magnetic bead samples, and functions to separate nucleicacid volumes from the set of nucleic acid-magnetic bead samples. StepS440 preferably reduces a concentration of unwanted matter from the setof biological samples being processed, to an acceptable level; however,Step S440 may alternatively entirely remove substantially all unwantedsubstances from the set of biological samples being processed. Step S440preferably includes providing a magnetic field S441, such that eachfluidic pathway in the set of fluidic pathways is configured to crossthe magnetic field. Preferably, the set of nucleic acid-magnetic beadsamples is captured and isolated within portions of the set of fluidicpathways crossing the magnetic field. Step S440 may further compriseproviding a heater configured to span a heating region of the set offluidic pathways S442, but may alternatively comprise providing multipleheaters or altogether omit providing a heater. In embodiments whereinmultiple heaters are provided, each heater is preferably independent toallow independent control of heating time and temperature for eachsample. Step S442 functions to provide a heater, which, in combinationwith a release solution that provides a a pH shift, facilitate a rapidand efficient unbinding of the nucleic acids from magnetic beads.

Step S440 may further comprise occluding at least one fluidic pathway ofthe set of fluidic pathways at a subset of a set of occlusion positionsS443 (and opening a previously occluded channel), which functions todefine at least one truncated fluidic pathway containing a nucleicacid-magnet bead sample and coupled to a source for delivery of a washsolution and a release solution. Preferably, Step S443 comprisesdefining at least one truncated fluidic pathway coupled to a wastechamber and to a fluid port, which functions to facilitate washing of atleast one nucleic acid-magnetic bead sample in the set of nucleicacid-magnetic bead samples, and releasing of at least one nucleic acidvolume from the set of nucleic acid-magnetic bead samples. Step S440 mayadditionally comprise delivering a wash solution through a portion of atleast one fluidic pathway S444, such as the truncated fluidic pathwaydefined in Step S443, and delivering a release solution through aportion of at least one fluidic pathway S445, such as the truncatedfluidic pathway defined in Step S443. Step S444 functions to wash atleast one nucleic acid-magnetic bead sample in the set of nucleicacid-magnetic bead samples, and Step S445 functions to release at leastone nucleic acid volume from the set of nucleic acid-magnetic beadsamples. The heater provided in Step S442 may be activated after StepS445 to induce a pH shift.

In a specific example of Step S440, the set of fluidic pathwayscontaining a set of nucleic acid-magnetic bead samples, from thespecific example of Step S430, is occluded at a subset of the set ofocclusion positions by a valve actuation subsystem of the moleculardiagnostic module, to define a set of truncated fluidic pathways coupledto a waste chamber and to a shared fluid port of the microfluidiccartridge for delivery of a wash solution and a release solution. Theliquid handling system delivers a wash fluid through the shared fluidport to wash the set of nucleic acid-magnetic bead samples, capturedwithin the magnetic field, and then delivers a release fluid through theshared fluid port to release a set of nucleic acid volumes from the setof nucleic acid-magnetic bead samples. In the specific example, eachfluidic pathway is washed sequentially, and the release solution isdelivered to each fluidic pathway sequentially to ensure that each laneis provided with substantially equal amounts of wash and releasesolutions. All waste fluid produced in the specific example of Step S440pass into the waste chamber coupled to the set of truncated fluidicpathways.

Step S450 recites combining each nucleic acid volume of the set ofnucleic acid volumes with a molecular diagnostic reagent of a set ofmolecular diagnostic reagents to produce a set of nucleic acid-reagentmixtures, which functions to prepare the set of nucleic acid volumes tobe detected. For each nucleic acid volume in the set of nucleic acidvolumes, Step S450 preferably comprises aspirating an entire volume ofthe nucleic acid volume from its corresponding fluidic pathway, andtransferring the nucleic acid volume to a well containing a moleculardiagnostic reagent. Preferably, all nucleic acid volumes in the set ofnucleic acid volumes are aspirated and combined with moleculardiagnostic reagents simultaneously using a multichannel fluid deliverysystem; however, each nucleic acid volume in the set of nucleic acidvolumes may alternatively be aspirated and combined with moleculardiagnostic reagents independently of the other nucleic acid volumes. Themolecular diagnostic reagents preferably comprise reagents configured toanalyze the set of nucleic acid volumes for markers of at least one ofgonorrhea (GC), Chlamydia (CT), herpes simplex virus (HSV), humanimmunodeficiency virus (HIV), human respiratory diseases, vaginaldiseases, gastrointestinal diseases due to bacterial and/or viralinfections (e.g., due to norovirus, rotavirus, adenovirus, sapovirus,astrovirus, etc.), hepatitis C virus (HCV), hepatitis B virus (HBV),trichonomas, human papillomavirus (HPV), group B Streptococcus (GBS),factor 2 (FII) gene, and factor five (FV) gene, but may alternativelycomprise reagents used to detect any specific nucleic acid sequenceassociated with other diseases and/or panels of agents.

In a first variation of Step S450 as shown in FIG. 16A, a nucleic acidvolume is aspirated and combined with a molecular diagnostic reagent fora single assay. In the first variation of Step S450, a set of nucleicacid volumes may thus be aspirated simultaneously, and each nucleic acidvolume may be transferred to an individual well to be combined with amolecular diagnostic reagent of a set of molecular diagnostic reagentsto produce a set of nucleic acid-reagent mixtures. In the firstvariation of Step S450, all nucleic acid-reagent mixtures in the set ofnucleic acid-reagent mixtures may or may not be substantially identicalin composition, depending on the homogeneity of the biological samplesused in Step S410; however, the first variation of S450 preferablycomprises using identical molecular diagnostic reagents, such thatidentical molecular diagnostic protocols analyzing identical markers maybe performed. Thus, the first variation of Step S450 encompasses runningmultiple identical tests from a stock biological sample (e.g., amultiplex assay), and running identical tests using a set ofsubstantially different biological samples (e.g., from differentsources).

In a second variation of Step S450, as shown in FIG. 16B, the set ofnucleic acid volumes is aspirated, and each nucleic acid volume in theset of nucleic acid volumes is combined with a molecular diagnosticreagent of a set of molecular diagnostic reagents. In the secondvariation of Step S450, the set of molecular diagnostic reagentspreferably comprises different molecular diagnostic reagents, such thatdifferent molecular diagnostic protocols analyzing different markers maybe performed. Thus, the second variation encompasses running multiplesubstantially different tests using a stock biological sample, andrunning substantially different tests using substantially differentbiological samples (e.g., from different sources).

In a specific example of Step S450, a multichannel liquid handlingsystem aspirates approximately 18 μL of each of a set of nucleic acidvolumes from the microfluidic cartridge used in the specific example ofStep S440 using a set of pipette tips, punctures at least one foil seal195 of at least one assay strip, wherein each well of the at least oneassay strip contains molecular diagnostic reagents, and dispenses eachaspirated nucleic acid volume into a well of the assay strip. In thespecific example of S450, the multichannel liquid handling system thenaspirates and dispenses the contents of each well approximately 10 timesto reconstitute molecular diagnostic reagents and mix the contents ofeach well.

Step S460 recites transferring each of the set of nucleic acid-reagentmixtures, through the corresponding fluidic pathway of the set offluidic pathways, to a detection chamber of a set of detection chambers,which functions to deliver the set of nucleic acid-reagent mixtures toan isolated detection chamber for further processing and analysis.Preferably, all nucleic acid-reagent mixtures in the set of nucleicacid-reagent mixtures are transferred simultaneously to the set offluidic pathways, but alternatively, each nucleic acid-reagent mixturein the set of nucleic acid reagent mixtures may be transferred to acorresponding fluidic pathway independently of the other nucleic acidreagent mixtures. Step S460 may further comprise occluding at least onefluidic pathway of the set of fluidic pathways at a subset of a set ofocclusion positions S462, which functions to define at least onetruncated fluidic pathway coupled to a detection chamber of a set ofdetection chambers. Preferably, Step S462 comprises occluding eachfluidic pathway of the set of fluidic pathways at a subset of a set ofocclusion positions, thus defining a set of truncated fluidic pathways,each coupled to a detection chamber.

In a specific example of Step S460, the multichannel liquid handlingsubsystem of the specific example of Step S450 transfers a set ofnucleic acid-reagent mixtures, each having a volume of approximately 16μL, back to the set of fluidic pathways of the microfluidic cartridge ofthe specific example of Step S450. Each nucleic acid-reagent mixture inthe set of nucleic acid-reagent mixtures is transferred at a rate of 50μL/minute. Manipulation of the elastomeric layer at a subset of a set ofocclusion positions by the valve actuation subsystem of the moleculardiagnostic module defines a set of truncated fluidic pathways, eachcoupled to a detection chamber, such that each nucleic acid-magneticbead sample in the set of nucleic acid-magnetic bead samples is isolatedwithin a truncated fluidic pathway of the set of truncated fluidicpathways. In the specific embodiment the occlusion position immediatelyupstream of the detection chamber and the occlusion position immediatelydownstream of the detection chamber are normally closed positions.During delivery, the multichannel liquid handling subsystem generatespressure to cause the elastomeric layer at the normally closed positionsto deform and allow fluid to flow through the normally closed positions.Once the pressure drops after the detection chamber is filled and themultichannel liquid handing subsystem ceases delivery, the elastomericlayer is configured to overcome the pressure in the channel andrecloses, thereby sealing the normally closed positions. The normallyclosed positions are then compressed using the valve actuation subsystemduring thermocycling to prevent pressures generated during a moleculardiagnostic assay to cause the normally closed positions to leak. Afterthe molecular diagnostic assay is complete and the occlusion “pins”withdrawn, the normally closed positions allow the samples and ampliconsto be trapped within detection chambers, substantially reducing the riskof contamination of the lab or other samples.

Step S470 recites receiving light from the set of nucleic acid-reagentmixtures, and functions to produce emission responses from the set ofnucleic acid-reagent mixtures in response to transmission of excitationwavelength light or chemiluminescent effects. Preferably, Step S470comprises the ability to transmit light including a wide range ofwavelengths through a set of excitation filters and through a set ofapertures configured to individually transmit light having single ormultiple excitation wavelengths onto the set of nucleic acid-reagentmixtures, and receiving light through a set of emission filters, fromthe set of nucleic acid-reagent mixtures. Step S470 may additionallycomprise reflecting light from the set of excitation filters off of aset of dichroic mirrors, and transmitting light through the set ofdichroic mirrors to a set of photodetectors. A specific example of StepS470 comprises using the optical subsystem 180 of the system 100described above to transmit and receive light; however, alternativevariations of Step S470 may use any appropriate optical systemconfigured to transmit light at excitation wavelengths toward the set ofnucleic acid-reagent mixtures, and to receive light at emissionwavelengths from the set of nucleic acid-reagent mixtures.

Step S480 recites generating a set of data based on light received fromthe set of nucleic acid-reagent mixtures, which functions to producequantitative and/or qualitative data from the set of nucleicacid-reagent mixtures. Step S480 may further function to enabledetection of a specific nucleic acid sequence from the nucleicacid-reagent mixture, in order to identify a specific nucleic acidsequence, gene, or organism. Preferably, Step S480 includes convertingelectrical signals, produced by a set of photodetectors upon receivinglight from the set of nucleic acid-reagent mixtures, into a quantifiablemetric; however, S480 may alternatively comprise convertingelectromagnetic energy, received by a set of photodetectors from the setof nucleic acid-reagent mixtures, into a set of qualitative data. In onevariation of Step S480, the set of data may be processed by a processorand rendered on a user interface; however, in other variations of StepS480, the set of data may alternatively not be rendered on a userinterface.

The method 400 may further comprise re-running a biological sample S490if processing and/or analysis of the biological sample results in lessthan ideal results. Preferably, Step S490 occurs if an analysis of abiological sample is indeterminate due to machine or user error.Additionally, Step S490 preferably occurs automatically upon detectionof a less than ideal result, but may alternatively occur in response toa user prompt. Block S490 is enabled due to rapid processing enabled byan embodiment of the system 100 described above, wherein rerunning asample without sample degradation is feasible during the time it takesto run a biological sample and determine if an analysis of thebiological sample has produced an indeterminate result.

2.1 Method—Additional Applications

As shown in FIG. 25A, a method 500 can include one or more of:processing a primary container containing a primary specimen of a sampleS510; returning outputs associated with one or more of system operationand sample analysis results S520; providing additional workflowoperation modes S530; performing error correction actions in associationwith a workflow error S540; implementing enhanced processing operationmodes S550; and performing other suitable operations in relation tosample processing workflows, output return, error correction, andsecurity. In an example workflow, the system can process one or moreprimary containers, such as a primary tube, or a secondary container(e.g., daughter container), transfer contents of the one or more primarycontainers (e.g., to be combined with a set of magnetic particles in acapture plate), and then transfer captured contents to a microfluidiccartridge for processing by a molecular diagnostic module.

In processing samples with units of the microfluidic cartridge, thesystem can cooperate with a processor in communication with acontroller, comprising a non-transitory computer-readable mediumcomprising instructions stored thereon, that when executed by theprocessor perform steps of automatically delivering a unit of thecartridge to the cartridge platform of the molecular diagnostic module,by way of the robotic arm (or other robotic module or roboticcomponent), processing one or more samples at the unit of the cartridgewithin the molecular diagnostic module, removing the unit of thecartridge from the molecular diagnostic module upon completion ofprocessing of the one or more samples (as described above), anddelivering a used unit of the cartridge, by way of the robotic arm (orother robotic module or robotic component), to a waste location of thesystem.

Blocks of the method 500 can be implemented in coordination with one ormore blocks of the method 400, in relation to pre-analytical,analytical, and/or post-analytical sample processing.

The method 500 functions to enable features, that during implementationimprove operational efficiency (e.g., in relation to continuous sampleprocessing, in relation to comparison of application data frameworks),editing of assay procedures, processing of results, controls, systemcalibration, security (e.g., firewalls, software virus mitigation,malware protection, remote monitoring, localization, IP configurations,etc.), and other system features (e.g., with respect to time zonechanges across different boot sessions, correction of networkpath-related errors, enhancement of boot options, methods of reportingtarget flags using Health Level 7 (HL7) standards, methods of correctingerrors associated with test orders for loaded samples, methods ofreporting results in a localized language, etc.).

The method 500 is preferably implemented by an embodiment, variation, orexample of system components described above, or can alternatively beimplemented using other system components. One or more portions of themethod 500 can be implemented, at least in part, by way of anon-transitory computer-readable medium comprising instructions storedthereon, that when executed on a processor perform steps of the method500. In one implementation, the method 500 can function to improveoperation of sample processing (e.g., pre-analytical, analytical,post-analytical process) with microfluidic elements, reagents (e.g.,reagents on-board a compact and automated system), liquid handlingsystem elements (e.g., with aspiration and delivery modes of operation),and containers (e.g., primary tubes).

Block S510 recites: processing a primary container containing a primaryspecimen of a sample, which functions to enable direct handling ofprimary tubes in an efficient manner and that reduces or otherwiseeliminates manual operation steps during sample processing. In relationto Block S510, the method 500 can be implemented using computer-readablemedium comprising instructions stored thereon, that when executed on aprocessor perform assignment of the type of container (e.g., tube type,such as plasma preparation tube, serum separation tube, etc.) of theprimary container containing the primary specimen, execution of modifiedhandling modes for low volume specimen containers (e.g., with mixvolume-based calculations based on detected specimen volumes),localization of specimen derived material and/or containers throughoutthe system, handling a wide range of aspiration volumes (e.g., from1-2000 uL), and other operational improvements. In variations, protocolsimplemented during performance of Block S510 can be associated withconfiguration files including one or more of: total aspirate anddispense monitoring (TADM) curves and bands (e.g., for various liquidclasses), extensible markup language (XML) files, applicationdevelopment framework (ADF) files, and other file/language formatsassociated with various labware definitions. Variations of Block S510can, however, be implemented in another suitable manner.

Block S520 recites: returning outputs associated with one or more ofsystem operation and sample analysis results, which functions to improvesystem operation, system control by an operator, and/or to provideinsights associated with sample processing in a manner that improvessample analysis. In embodiments, Block S520 can include method steps forincreasing or reducing the amount of information returned to an operatoror other entity (e.g., in relation to passing data or content betweensystem components involved in sample processing), providinglocalization-related information, improving returning of resultsassociated with sample filtering, improving returning of results inrelation to exporting of content, and otherwise improving consistency,precision, and accuracy of returned outputs.

In variations, Block S520 can include one or more of: omitting reportingof negative results (e.g., reporting of Ct values, reporting of othervalues, etc.) in returned reports associated with sample analysis,providing enhanced outputs with localization information, providinginformation in an accurate manner with the ability for an operator tofilter by sample (or other sample-related factors), providingfunctionality for exporting returned outputs in a variety of formats,returning specific outputs (e.g., log concentration, etc.), improvingconsistency of returned outputs (e.g., with respect to rounding ofvalues, etc.), providing the ability for an operator to select whichtest results to include in a summary report (e.g., with organization bytime, by assay type, etc.), hiding less relevant report aspects (e.g.,log of concentration for a qualitative assay, etc.), providingrecommendations (e.g., to dilute samples for quantitative results abovea threshold or saturation level, recommendations in relation to peakminimum cycles, etc.), reporting use of an un-preferred buffer, andother reporting aspects. Other variations of returning outputs can,however, be implemented in another suitable manner.

Block S530 recites: providing additional workflow operation modes, whichfunctions to provide enhanced modes of system operation. In embodiments,Block S530 can provide functionality for processing samples (e.g., usingcustom tests, using lab developed tests (LDT), etc.), executing storagemodes of operation (e.g., for sample storage, for reagent storage,etc.), adjusting processing for various sample parameters (e.g., samplevolume, number of mixing step instances, number of washing stepinstances, etc.), providing monitoring of sample container statuses, andperforming other workflow operation modes. In variations, Block S530 caninclude one or more of: providing extraction-only modes of operation(e.g., in a lab developed test), performing operations on a single teststrip (e.g., in a lab developed test), implementing operation modes uponanalysis of process material codes (e.g., buffer product codes, stripproduct codes, etc.), performing automated maintenance (e.g., on aweekly schedule, on another schedule), performing automatic restartmodes of operation (e.g., in response to errors or other states),enabling entering of a long-term storage mode of operation, providingfunctionality for adjusting specimen mix volume and number of mixes(along with other mixing parameters), providing functionality forreporting statuses of specimen processing containers and samplemonitoring, providing functionality for unloading and/or reloading ofcarriers through the service door workflow, providing functionality forfiltering system events by error code, providing functionality forprocessing extended barcode characters, providing functionality for bulkreagent replacement (e.g., when there are no samples being processed,when samples are being processed, etc.), providing functionality fordisplaying number of samples loaded/selected, providing functionalityfor implementing changes to specimen processing under control of asupervising entity, providing functionality for unlocking extractionmodule apparatus in the event of a dropped sample container, providingfunctionality for grouping specimen types with user-defined controls,implementing workflows for calibration of multiple molecular diagnosticmodules, performing cleaning operation modes, performing preventativemaintenance tracking, providing a system manifest, providingfunctionality in multiple languages, and performing other operations.

Block S540 recites: performing error correction actions in associationwith a workflow error, which functions to improve system operation inrelation to undesired system behavior. In embodiments, Block S540 caninclude performing error correction actions in response to one or moreof: sample processing workflow errors/undesired operation, general userinterface (GUI) errors/undesired operation, hardware interfaceerrors/undesired operation, and other types of errors/undesiredoperation.

In variations associated with sample processing workflowerrors/undesired operation, Block S540 can include one or more of:performing an error correction action in response to sample carrierloading issues from user loading, performing an error correction actionin response to unnecessary delays in sample processing, performing anerror correction action in response to issues during sample loading(e.g., by generating and executing control instructions to pause and/oreject a sample container), modifying scheduling (e.g., controlscheduling behavior during error handling or other situations in amanner that does not adversely affect throughput, optimizing schedulingin relation to release buffer heating requirements, returning improvedestimates of completion times, etc.), improving performance of LHPC1associated methods, performing an error correction action in response tosamples stuck in a phase of a PCR process (e.g., by generating andexecuting control instructions to pause sample movement, heating,cooling, or other aspects, by generating and executing controlinstructions to eject a sample container, etc.), performing an errorcorrection action in response to samples stuck in a phase of anextraction process (e.g., by generating and executing controlinstructions to pause sample movement, aspiration, operation of a fluidhandling subsystem, or other aspects, by generating and executingcontrol instructions to eject a sample container, etc.), performing anerror correction action in response to sample stuck during a phase ofcartridge loading or a sample of the set of samples failing to proceedto a next step of sample processing (e.g., by generating and executingcontrol instructions to pause sample movement and/or to eject a samplecontainer, etc.), performing an error correction action in response toerrors experience during test order importing (e.g., applicationexceptions, cosmetic issues, etc.), re-running sample controls inresponse to an unresolved result (e.g., with assay specific settingsand/or global settings), performing an error correction action inresponse to database failure issues, performing an error correctionaction in response to a priming frequency issue, performing actions toprevent leakage during washing, allowing an operator to cancelindividual in-process tests, preventing an operator from changing samplecontainer settings for calibration and/or external control factors, andperforming other suitable response actions in response to workflowerrors.

In variations associated with GUI errors/undesired operation, Block S540can include one or more of: generating user permissions for databaserelated operations (e.g., database restoration, database export,database modification, etc.), returning notifications in response tofailures in data integrity, performing an error correction action inresponse to changes in the numbers of sample containers processed (e.g.,in relation to container counting), performing an error correctionaction in relation to molecular diagnostic system access modes (e.g., inrelation to service door access and operation, reagent drawer operation,waste drawer operation, and system time out behavior, etc.), performingan error correction action in relation to reporting of waste binemptying status, correcting errors associated with applicationdevelopment framework aspects (e.g., importation, market assignment,etc.), adjusting references between molecular diagnostic system elements(e.g., from internal control to sample process control, from heatermodule to extraction plate, etc.), performing baseline corrections bysingle point (e.g., fluorescence at fixed baseline start cycle withnormalized data), and performing other suitable response actions inresponse to GUI errors (e.g., errors in providing warnings, non-idealwording of reports, formatting of tables, localization, providingviewing of reports, providing functionality for network mapping, etc.).

In variations associated with hardware interface errors/undesiredoperation, Block S540 can include one or more of: providing indicationsassociated with proper or improper system operation (e.g., visualindicator light operation modes with respect to sample container loadingand other system states), executing calibration-associated configurationsettings and memory-associated operations, performing error correctionactions in response to bar code reading errors (e.g., in relation tore-scanning of a bar code, in relation to providing notifications, inrelation to ejecting of an improperly bar coded sample container, etc.),performing calibration-associated operations with respect to the valveactuation subsystem 170 described above (e.g., with respect tocam-component position modification after calibration, by re-positioninga valve actuation subsystem 170 component relative to cartridge 110post-calibration, by performing a valve actuator calibration operationfor a valve actuation subsystem of the molecular diagnostic module,wherein the valve actuator calibration operation guides re-positioningof a cam of the valve actuation subsystem relative to the cartridge,etc.), performing calibration-associated operations with respect toactuator (e.g., jack system) components (e.g., with respect to homingand other operations, by performing a cartridge platform actuatorcalibration operation, wherein the cartridge platform actuatorcalibration operation guides re-homing of a cartridge platform actuatorin communication with the cartridge platform), and performing otheractions associated with hardware errors.

Block S550 recites: implementing enhanced processing operation modes,which functions to improve system operation and sample processing duringcalibration, sample processing, and other pre-analytical, analytical,and/or post-analytical phases of operation. In variations, Block S540can include one or more of: applying a stored standard curve forquantitation of different sample types with different aspiration volumesin order to provide enhanced quantitative calibration factors forstreamlined analyses, performing an inhibition check for qualitativeassays (e.g., by calling a non-amplified target unresolved if a sampleprocess control is also not amplified and another target is amplified ata non-SPC competitive level) in a manner that reduces false negativerisks for co-infection with inhibition, performing an electronparamagnetic resonance (EPR) check failed result (e.g., in a manner thatcan be set in the application development framework, as an indeterminateor negative result), implementing a quantitative dilution factor (e.g.,from 1:1 to 1:10), implementing standard curve improvements (e.g., withadjustment of an intercept range from 10-200, etc.), and performingother processing operation modes. In particular, an unresolved resultcan be a result matching predefined criteria by a processor of thesystem, and the non-transitory computer-readable medium can includeinstructions for automatically re-processing a sample of the set ofsamples in response to return of a result matching predefined criteriaby the processor. In examples, the predefined criteria can be used tore-run a sample due to return of an invalid result, and/or can be usedto run a follow up test in response to a returned result that warrantsadditional assessment.

In more detail, the non-transitory computer-readable medium can furtherinclude instructions for using a stored calibration equation derived forone specimen volume, and returning a quantitation value for a range ofspecimen values, by implementing a calibration correction factor and thestored calibration equation, upon processing a sample with thecartridge. Additionally or alternatively, the non-transitorycomputer-readable medium can further include instructions for processinga dilution of the sample with implementation of a dilution adjustmentfactor (e.g., determined for one of a range of dilutions) and the storedcalibration equation, and automatically reporting a target quantitationvalue for the sample prior to the dilution. In association with returnof a target quantitation value, the non-transitory computer-readablemedium can further include instructions for one or more of:automatically applying a qualification flag onto the target quantitationvalue if a set of criteria for a sample process control is unmet (e.g.,if there is delayed amplication or other issues with the sample processcontrol), and entering a quantitation check mode of operation inresponse to the qualification flag; performing a correction operation,with application of a correction factor derived from a set of parametersattributed to amplification of the sample process control, and returninga corrected value of the target quantitation value; and for amulti-target assay, entering an inhibition check mode of operation inresponse to return of the target quantitation value for a single targetof a set of targets (e.g., which could indicate inhibition of one ormore targets of the multi-target assay).

In one implementation, as shown in FIG. 25B, one or more portions of themethod 500 can be implemented in coordination with an embodiment,variation, or example of the system(s) described above and/or shown inrelation to FIGS. 1A-23, and include: receiving a cartridge at thecartridge platform S501; positioning a heater at the first surface ofthe cartridge S502; executing a reagent receiving mode with movement ofan aspiration and delivery head of a fluid handling system into fluidcontact with at least one storage container S503; executing a reagentdelivery mode with movement of the aspiration and delivery head of thefluid handling system into contact with a port of the cartridge S504,and performing an inventory management operation, where the inventorymanagement can involve movement of one or more of: the cartridge and theone or more reagents, and/or be performed in response to entrance of thereagent receiving mode, entrance of the reagent delivery mode, andreception of the cartridge S505. The inventory management operation caninclude one or more of automatically adding new materials (e.g.,cartridges, containers, reagents, etc.) to the system and/or disposingof used or expired materials (e.g., cartridges, containers, reagents,etc.), in coordination with other subsystems (e.g., the fluid handlingsubsystem).

Alternatively, performance of the inventory management operation can beperformed in another suitable manner (e.g., based on another triggeringevent). For instance, the non-transitory computer-readable mediumdescribed above can include instructions for monitoring a useful life ofeach unit of the cartridge associated with the system, and automaticallyremoving, by way of the robotic arm (or other robotic module or roboticcomponent), one or more units of the cartridge surpassing the usefullife (e.g., using a grabbing function of the robotic arm (or otherrobotic module or robotic component) to an expired cartridge location ofthe system. Additionally or alternatively, the non-transitorycomputer-readable medium can include instructions for performing aninventory management operation ahead of performance of the moleculardiagnostic reaction, thereby promoting continuous and efficientoperation of the system.

In particular, the inventory management operation and other operationsare configured to promote smooth and efficient operation of the system.As such, the non-transitory computer-readable medium of the system caninclude instructions for delivering the one or more samples to one ormore units of the cartridge in a continuous operation mode, wherein thecontinuous operation mode minimizes interruptions between instances ofsample delivery to the one or more units of the cartridge.

Furthermore, the portion of the method 500 shown in FIG. 25B can adaptedto include one or more steps of the method(s) described above.

Other methods can include steps for executing one or more of: automaticlighting system calibration, improvement of diagnostic service and busaspects to support various protocols, addition of guard bands (e.g., fortarget material release, magnet heating, lysis heating, etc.),application of bug fixes, improvement of extraction board aspects (e.g.,cable insert changes), and/or other system enhancements.

In related embodiments, an alternative method can include: receiving aunit of the cartridge at a cartridge platform of a molecular diagnosticmodule cooperating with a fluid handling subsystem comprising anactuator and an aspiration and delivery head coupled to the actuator,and a reagent receptacle comprising a set of storage containers for aset of reagents stored in an ambient condition (and/or with reagentscooled to below the ambient condition); and executing a reagentreceiving mode with movement of the aspiration and delivery head of thefluid handling system into fluid contact with at least one of the set ofstorage containers; and executing a reagent delivery mode with movementof the aspiration and delivery head of the fluid handling system intocontact with a port (e.g., inlet or outlet) of the unit of thecartridge.

In related embodiments, an alternative method can include receiving aset of containers (e.g., primary containers, secondary containers, etc.)at a sample processing system comprising a reagent receptacle comprisinga set of storage containers for a set of reagents stored in an ambientcondition, a fluid handling subsystem comprising an actuator and anaspiration and delivery head coupled to the actuator, and a moleculardiagnostic module; transferring, using the fluid handling subsystem,material of one or more of the set of containers to the moleculardiagnostic module for processing; in coordination with transferringmaterial of each of the set of containers to the molecular diagnosticmodule: executing a reagent receiving mode with movement of theaspiration and delivery head of the fluid handling system into fluidcontact with at least one of the set of storage containers; andexecuting a reagent delivery mode with movement of the aspiration anddelivery head of the fluid handling system into communication with themolecular diagnostic module.

In related embodiments, an alternative method can include: receiving aset of containers (e.g., primary containers, secondary containers,etc.), containing a set of samples, at a sample processing systemcomprising a reagent receptacle comprising a set of storage containersfor a set of reagents stored in an ambient condition, a fluid handlingsubsystem comprising an actuator and an aspiration and delivery headcoupled to the actuator, and a molecular diagnostic module; generating asample processing regimen based upon an analysis of at least one of: aset of characteristics of the set of samples and a set ofoperator-defined constraints; and processing the set of samplesaccording to the sample processing regimen. In particular, the analysiscan be based upon one or more of: urgency of acquiring sample-derivedresults, as defined by at least one of: the set of operator-definedconstraints and a set of constraints automatically defined in logic ofthe sample processing system; an optimization for maximum throughput ofsample processing, based upon the set of characteristics of the set ofsamples; an optimization for efficacy of sample processing, based uponthe set of characteristics of the set of samples, wherein the set ofcharacteristics comprises a sample stability factor; an optimization forminimization of reagent waste, based upon the set of characteristics ofthe set of reagents, wherein the set of characteristics comprises areagent expiration factor; sample type, test type, RNA sampleprocessing, and DNA sample processing; and/or any other suitablecriteria. As such, the system can take inputs and/or process built-inlogic to prioritize processing of samples in an efficient manner, basedon expiration factors for containers and/or reagents, and/or otherfactors. In embodiments, generation of the analysis can be based uponanalytical methods involving decision trees, rule-based approaches,and/or other approaches.

In related embodiments, an alternative method can include: receiving aset of containers (e.g., primary containers, secondary containers,etc.), containing a set of samples, at a sample processing systemcomprising a reagent receptacle comprising a set of storage containersfor a set of reagents stored in an ambient condition, a fluid handlingsubsystem comprising an actuator and an aspiration and delivery headcoupled to the actuator, and a molecular diagnostic module; generating aprocessing regimen based upon an analysis of a set of characteristics ofthe set of reagents and the set of containers; and processing the set ofsamples according to the processing regimen. In particular, the set ofcontainers and the set of reagents are each produced using batchproduction processes and each contain more than one unitized reaction,and are configured to be implemented in a unitized operation.

Embodiments of the methods and variations thereof can be embodied and/orimplemented at least in part by a machine configured to receive acomputer-readable medium storing computer-readable instructions. Theinstructions are preferably executed by computer-executable componentspreferably integrated with the system 100 and one or more portions ofthe processor 273 and/or the controller 272. The computer-readablemedium can be stored on any suitable computer-readable media such asRAMs, ROMs, flash memory, EEPROMs, optical devices (CD or DVD), harddrives, floppy drives, or any suitable device. The computer-executablecomponent is preferably a general or application specific processor, butany suitable dedicated hardware or hardware/firmware combination devicecan alternatively or additionally execute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of systems, methods and computer programproducts according to preferred embodiments, example configurations, andvariations thereof. In this regard, each block in the flowchart or blockdiagrams may represent a module, segment, or portion of code, whichcomprises one or more executable instructions for implementing thespecified logical function(s). It should also be noted that, in somealternative implementations, the functions noted in the block can occurout of the order noted in the FIGURES. For example, two blocks shown insuccession may, in fact, be executed substantially concurrently, or theblocks may sometimes be executed in the reverse order, depending uponthe functionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts, or combinations of special purpose hardware andcomputer instructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

What is claimed is:
 1. A system for automatically processing nucleicacid content of one or more samples in cooperation with a cartridgecomprising a set of microfluidic pathways for performing amplificationand detection of said nucleic acid content, the system comprising: Oneor more lysis containers for executing a lysis operation on one or moresamples and capturing nucleic acid content resulting from the lysisoperation at a set of beads coupled with chemical entities for capturingsaid nucleic acid content; a molecular diagnostic module comprising: acartridge location for positioning the cartridge within the moleculardiagnostic module, one or more heaters at the cartridge location foramplifying said nucleic acid content, and an optical subsystem fordetecting said nucleic acid content; one or more storage containers forone or more reagents, the one or more reagents comprising: a releasesolution for releasing at least a portion of the nucleic acid contentfrom the set of beads, and reagents for amplifying one or more nucleicacids; a fluid handling subsystem comprising an actuator and anaspiration and delivery component; and one or more processors comprisinga non-transitory computer-readable medium comprising instructions storedthereon, that when executed by the one or more processors perform stepsof: positioning the cartridge at the cartridge location, executing adelivery mode with movement of the aspiration and delivery component ofthe fluid handling system between the one or more lysis containers, theone or more storage containers, and one or more inlets of the cartridge,thereby delivering said nucleic acid content into the cartridge,transmitting heat to one or more amplification regions of the set ofmicrofluidic pathways of the cartridge with the one or more heaters,thereby performing amplification of said nucleic acid content, detectingthe nucleic acid content with the optical subsystem, and delivering thecartridge from the cartridge location.
 2. The system of claim 1, whereinthe set of beads comprises at least one of magnetic beads andparamagnetic beads.
 3. The system of claim 1, further comprising one ormore magnets positioned proximal to at least one of the one or morelysis containers and the cartridge for retention of the set of beads inposition.
 4. The system of claim 3, wherein the one or more magnetscomprises at least one of a permanent magnet and an electromagnet. 5.The system of claim 3, wherein the one or more magnets comprises a setof magnets, each subset of magnets in the set of magnets coupled to andseparated from an adjacent subset of magnets with each subset of magnetsdedicated to a sample of the one or more samples.
 6. The system of claim1, wherein the molecular diagnostic module further comprises a valveactuation subsystem for directing fluid flow within the cartridge. 7.The system of claim 6, wherein the non-transitory computer-readablemedium further comprises instructions stored thereon, that when executedby the one or more processors perform: transitioning one or more of theset valves to between a first state and a second state, and incoordination with the fluid handling system, transmitting said nucleicacid content downstream within the set of microfluidic pathways prior toamplification.
 8. The system of claim 1, wherein performingamplification and detection of said nucleic acid content comprisesperforming a real time PCR reaction.
 9. The system of claim 1, whereinthe cartridge location comprises a platform aligned with the one or moreheaters, the molecular diagnostic module further comprising an actuationsubsystem configured to displace at least one of the platform and theone or more heaters for relative displacement of the cartridge with theone or more heaters.
 10. The system of claim 9, wherein the opticalsubsystem is coupled to the actuation subsystem, the system comprising acartridge retention mode in which the actuation subsystem simultaneouslypositions the optical subsystem and the one or more heaters forcommunication with the cartridge at the cartridge location.
 11. A systemfor automatically processing nucleic acid content of one or more samplesin cooperation with a cartridge comprising a set of microfluidicpathways for performing amplification and detection of said nucleic acidcontent, the system comprising: one or more process containers forcapturing nucleic acid content from one or more samples at a set ofbeads coupled with chemical entities for capturing said nucleic acidcontent; a molecular diagnostic module comprising: a cartridge locationfor positioning the cartridge within the molecular diagnostic module,one or more heaters at the cartridge location for amplifying saidnucleic acid content, and an optical subsystem for detecting saidnucleic acid content; one or more storage containers for one or morereagents, the one or more reagents comprising a release solution forreleasing at least a portion of the nucleic acid content from the set ofbeads, and reagents for amplifying one or more nucleic acids; a fluidhandling subsystem comprising an actuator and an aspiration and deliverycomponent coupled to the actuator; and one or more processors comprisinga non-transitory computer-readable medium comprising instructions storedthereon, that when executed by the one or more processors perform stepsof: positioning the cartridge at the cartridge location, executing adelivery mode with movement of the aspiration and delivery component ofthe fluid handling system between the one or more storage containers,and one or more inlets of the cartridge, thereby delivering said nucleicacid content into the cartridge, transmitting heat to one or more of theset of microfluidic pathways of the cartridge with the one or moreheaters, thereby performing amplification and detection of one or morenucleic acids, and delivering the cartridge from the cartridge location.12. The system of claim 11, further comprising the cartridge, whereinthe cartridge comprises: a first layer, an intermediate substratecoupled to the first layer and defining the set of microfluidicpathways, and a region defining a set of valve positions for the set ofmicrofluidic pathways, wherein one or more of the set of valve positionsis transitionable between an occluded state and an open state.
 13. Thesystem of claim 12, wherein the molecular diagnostic module furthercomprises a valve actuation subsystem aligned with the cartridgelocation and configured to transition one or more of the set of valvepositions between the occluded state and the open state.
 14. The systemof claim 11, wherein the non-transitory computer-readable medium furthercomprises instructions stored thereon, that when executed by the one ormore processors perform: in coordination with the delivery mode,executing a lysis operation with delivery of a lysing reagent to one ormore samples retained at the one or more lysis containers by theaspiration and delivery head of the fluid handling system, and capturingnucleic acid content resulting from the lysis operation, at the set ofbeads within the one or more lysis containers.
 15. The system of claim14, wherein the non-transitory computer-readable medium furthercomprises instructions stored thereon, that when executed by the one ormore processors perform: retaining said nucleic acid content within theone or more lysis containers by way of the set of beads and one or moremagnets proximal the one or more lysis containers, and inducing a pHshift within the one or more lysis containers, with delivery of therelease solution into the one or more lysis containers, therebyreleasing said nucleic acid content from the set of beads.
 16. Thesystem of claim 11, wherein the set of beads comprises at least one ofmagnetic beads and paramagnetic beads.
 17. The system of claim 15,wherein the one or more magnets comprises a permanent magnet.
 18. Thesystem of claim 15, wherein the one or more magnets comprises a set ofmagnets, each magnet in the set of magnets coupled to and separated froman adjacent magnet by one of a set of magnet heating elements.
 19. Thesystem of claim 11, wherein performing amplification and detection ofsaid nucleic acid content comprises performing a real time PCR reaction.20. The system of claim 11, wherein the cartridge location comprises aplatform aligned with the one or more heaters, the molecular diagnosticmodule further comprising an actuation subsystem configured to displaceleast one of the platform and the one or more heaters for relativedisplacement of the cartridge with the one or more heaters.
 21. Thesystem of claim 20, wherein the optical subsystem is coupled to anactuator of the actuation subsystem, the system comprising a cartridgeretention mode in the actuator is extended to displace the platform, thecartridge at the platform is biased against the one or more heaters at afirst side of the cartridge, and the optical subsystem, coupled to theactuator is positioned at a second side of the cartridge.